WO2022035860A2 - Replication-competent adenovirus type 4-hiv env vaccines and their use - Google Patents

Abstract

Replication-competent adenovirus type 4 (Ad4)-based vectors expressing modified forms of human immunodeficiency virus (HIV) envelope (Env), and immunogenic compositions thereof, are described. An extensive array of modified Env proteins was generated and characterized for antigenicity and immunogenicity to identify recombinant Env proteins having a native-like conformation. Based on these studies, two Env vaccine candidates (Ad4-Env145NFL and Ad4-Env150KN) were selected for clinical studies. The recombinant Ad4-based HIV Env vectors can be used for preventing or inhibiting infection with HIV.

Description

REPLICATION-COMPETENT ADENOVIRUS TYPE 4-HIV ENV VACCINES

AND THEIR USE

CROSS REFERENCE TO RELATED APPLCATIONS

This application claims the benefit of U.S. Provisional Application No. 63/063,810, filed August 10, 2020, which is herein incorporated by reference in its entirety.

FIELD

This disclosure concerns modified human immunodeficiency virus (HIV)-l envelope (Env) polypeptides derived from clade C strain 1086. This disclosure further concerns replication- competent adenovirus type 4 (Ad4)-based vectors expressing the modified Env polypeptides and use of the Env-expressing adenoviruses as immunogenic compositions for preventing or inhibiting infection with HIV-1.

BACKGROUND

Three HIV vaccine candidates studied in large efficacy trials, two based on the use of recombinant bivalent HIV Env gpl20 (AIDSVAX B/B and B/E) and the third (“STEP” study) based on the use of a replication-deficient Ad5-vectored vaccine encoding Gag, Pol, and Nef antigens (Buchbinder et al., Lancet 372(9653) : 1881- 1893 , 2008; Pitisuttithum et al., J Infect Dis 194(12) : 1661- 1671 , 2006; Flynn et al., J Infect Dis 191(5):654-665, 2005), failed. The results of these trials suggested that protecting against HIV infection or suppressing viral replication following HIV infection were goals that would be difficult to achieve in humans. However, several important advances have generated considerable optimism that these goals may be achievable.

First, an HIV vaccine trial in Thailand demonstrated 30% overall efficacy in preventing infection in human participants who were primarily at heterosexual risk of HIV infection (Rerks- Ngarm et al., N Engl J Med 361(23): 2209-2220, 2009). In that trial, vaccinees were primed with a replication-defective canarypox vector expressing Gag, Pro, and Env, and boosted with Env gpl20 protein. Although the precise mechanism of action remains incompletely understood, protection from infection is believed to operate through an antibody-mediated mechanism. This study provided an important proof-of-concept that the humoral immune response can provide protection from infection with relatively diverse HIV isolates in humans.

A second set of observations was provided by a study demonstrating that the CD8+ T cell response may be used to suppress or possibly prevent lentiviral infection (Hansen et al. , Nature 473(7348):523-527, 2011). In that study, rhesus macaques were immunized with a rhesus cytomegalovirus (CMV) vaccine that encoded HIV gene products that are targets of the cellular immune response. This replication-competent vaccine induced a high-frequency simian immunodeficiency virus (SlV)-specific CD8+ T cell response. In animals that were infected, a profound level of suppression of the pathogenic SIV challenge virus was observed. Thus, in addition to an Env-specific humoral immune response to an HIV vaccine, the CD8+ T cell response may provide an important second line of defense against breakthrough infections. The most robust vaccine-induced restriction of viral replication has been generated by attenuated SIV or replication- competent rhesus CMV vectors. Similar observations have been made for vaccines against other viral infections in humans. Live replication-competent vectors are among the most immunogenic vaccines, but have not been extensively explored for HIV.

In a later Phase 1 trial, participants were given three immunizations with one or both of Ad4-mgag and Ad4-EnvC150 vaccines at months 0, 2, and 6 followed by the AIDSVAX B/E boost at month 8. The adenovirus-based vaccines were administered either by an enteric-coated capsule (delivered orally) or by intranasal spray. The results demonstrated low frequencies of HIV-specific CD4⁺ or CD8⁺ T cells and serum binding, but not neutralizing, antibodies. This finding is in contrast to the neutralizing antibodies induced by the Ad4-H5-Vtn vaccine administered to the upper respiratory tract in previous studies (Matsuda et al. , Sci Immunol 4(34): eaau2710, 2019). In that study, one vaccination induced persistent neutralizing antibodies with affinity maturation over 6 to 12 months. These antibodies were readily boosted by recombinant protein or split vaccines (Matsuda et al., J Clin Invest 131(5):el40794, 2021). Upon re-examination, it was determined that the cleavage incompetent 1086mutC Env protein expressed by the Ad4-EnvC150 vector was not in a native- like conformation, a feature thought to be essential for the induction of neutralizing antibodies (Ringe et al., Proc Natl Acad Sci USA 110(45): 18256-18261 , 2013).

Thus, there remains an urgent need for a safe and effective HIV vaccine.

SUMMARY

The present disclosure characterizes multiple HIV Env designs to identify recombinant Env proteins having a native-like conformation. An extensive array of modified Env proteins was generated and the impact of Env design changes on antigenicity and immunogenicity was examined. Based on these studies, two Env polypeptides (Envl45NFL and Envl50KN) were selected for further study. Recombinant, replication competent serotype 4 adenoviruses (Ad4) expressing the modified Env polypeptides were also developed. Ad4-Envl45NFL expresses stabilized and homogenous Env proteins, while Ad4-Envl50KN expresses non- stabilized and less uniform Env proteins. Modified Env polypeptides based on HIV-1 clade C, strain 1086 (“1086C”) are disclosed. In some embodiments, the modified Env polypeptide includes a heterologous CD5 signal peptide sequence at the N-terminus; a plurality of amino acid substitutions to stabilize Env trimer formation; an asparagine substitution at position 160; a proline substitution at position 559; a first heterologous peptide linker positioned between the gpl20 and gp41 subunits; a second heterologous peptide linker positioned between the ectodomain and the transmembrane domain; and a C-terminal truncation that results in deletion of the cytoplasmic domain, wherein the amino acid positions are numbered with reference to the HXB2 numbering scheme. In one non-limiting example, the modified Env polypeptide includes the amino acid sequence of SEQ ID NO: 3 (Envl45NFL).

In other embodiments, the modified Env polypeptide includes a native HIV-1 signal peptide at the N-terminus; an asparagine substitution at position 160, numbered with reference to the HXB2 numbering scheme; a cleavage competent sequence positioned between the gpl20 and gp41 subunits; and a C-terminal truncation that results in deletion of a portion of the cytoplasmic domain. In one non-limiting example, the modified Env polypeptide includes the amino acid sequence of SEQ ID NO: 6 (Envl50KN).

Env trimers comprised of the modified Env polypeptides disclosed herein are also provided. Also provided herein are recombinant adenoviruses that express a modified Env polypeptide. In some embodiments, the adenovirus is a replication competent adenovirus, such as replication competent Ad4. In specific non-limiting embodiments, the adenovirus genome includes the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

Further provided are nucleic acid molecules encoding a modified Env polypeptide disclosed herein. In specific non-limiting examples, the nucleic acid molecules include the sequence of SEQ ID NO: 2 or SEQ ID NO: 5. Vectors that include a disclosed nucleic acid are also provided. In some embodiments, the vector is an adenovirus vector, such as a replication competent adenovirus vector, for example a replication competent Ad4. In particular non-limiting examples, the vector includes the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

Also provided are immunogenic compositions that include a modified Env polypeptide, Env trimer, recombinant adenovirus, nucleic acid molecule, or vector disclosed herein and a pharmaceutically acceptable carrier.

Further provided is a method of eliciting an immune response against HIV-1 in a subject by administering to the subject an effective amount of a modified Env polypeptide, Env trimer, recombinant adenovirus, nucleic acid molecule, vector or immunogenic composition disclosed herein. In some embodiments, administration is via an intranasal or oral route. The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C: Env truncation to gpl45 increases cell surface expression of non-stabilized and SOSIP-stabilized Env. (FIG. 1A) Genetic maps of truncated Env designs. SOSIP stabilized designs are shown. (FIG. IB) Env expression was measured by the VRC01 median fluorescence intensity (MFI) normalized to the VRC01 MFI of the non-stabilized gpl60. (FIG. 1C) Truncation of gp41 had limited effects on Env conformation. Antibody MFIs were normalized to the VRC01 MFI of each construct to account for differences in Env expression. Broadly neutralizing antibodies (bnAbs) that demonstrate a native-like Env conformation are PGT145, PG16 and PGT151 and antibodies that bind CD4-inducible epitopes are F105 and 447-52D.

FIG. 2: Genetic maps of Env designs. Lengths are not to scale. MPER, membrane proximal external region; TM, transmembrane; DS, disulfide bond (see Table 2 for additional abbreviations).

FIG. 3: Antigenicity of Env designs expressed by the plasmid shuttle vector. MFI values were normalized to the VRC01 MFI of each construct to account for differences in Env expression.

FIGS. 4A-4B: Antigenicity of Env designs expressed by the Ad4 vector. (FIG. 4 A) Representative flow plots are shown for three Ad4-Env recombinants. (FIG. 4B) MFI values were normalized to the VRC01 MFI of each construct to account for differences in Env expression.

FIG. 5: Negative stain electron microscopy images of 3BNC117-bound Env. Representative images show large unstructured aggregates due to membrane extraction. Three representative class averages show Env formed correctly assembled trimers (*), dimers of trimers (2x), and trimers of trimers (3x). Image scale bar = 50 nm. 2D class average scale bar = 10 nm.

FIG. 6: Immunogenicity of Ad4-Env recombinants in rabbits. Rabbits were immunized at weeks 0 and 4. Sera was collected at weeks 0, 4, 8, and 12. Serum neutralization was tested by a pseudovirus entry inhibition assay against strains SF162 and 1086C.

FIG. 7: Rabbits were immunized on weeks 0 and 4 with Ad4-Env recombinants. Rabbits were boosted with the BG505 DS-SOSIP soluble trimer (VRC-HIVRGP09g-0 Trimer 4571) on week 12 and the 16055 Del Gly4 soluble trimer on week 20. Sera was collected at weeks 0, 4, 8, 12, 14, 16, 20, 22, 24, and 28. Serum neutralization was tested by a pseudovirus entry inhibition assay against strains SF162 and 1086C. Lines connect median values at each time point.

FIG. 8: Schematic illustrating non-stabilized and stabilized Env immunogens. FIG. 9: Genetic maps of Ad4-Env recombinants. Various Env genes were inserted within the Ad4 E3 region, which was either fully deleted or partially deleted. The Env gene was also inserted after the end of the partially deleted E3 region. Gene lengths are not to scale.

FIGS. 10A-10D: Gating strategy to generate MFI values. (FIG. 10A) Env and Hexon expressing cells were gated by forward scatter area (FSC-A) vs. side scatter area (SSC-A). (FIG. 10B) Dead cells were excluded by live/dead fixable violet dead cell stain vs. SSC-A. (FIG. 10C) Single cells were gated by forward scatter width (FSC-W) by forward scatter height (FSC-H). (FIG. 10D) MFI was calculated from cells positive for both Env and Hexon.

FIG. 11: Down- selection of stabilized Env designs by EEISA. Two stabilized Env designs were down-selected by a cell surface membrane protein ELISA. Env designs were ranked by CAP256 VRC26.25 times PGT145 values. Env designs with 17b, 17b with CD4, 447-52D, and F105 values above 0.2 were excluded.

FIG. 12: Down-selection of stabilized Env designs by flow cytometric analysis. Five stabilized Env designs were down-selected by flow cytometric analysis of PGT151, F105, 447- 52D, and VRC01 binding. MFI values were normalized to VRC01 MFI of each construct to account for differences in Env expression. Env expression is shown by the VRC01 MFI.

FIG. 13: Breadth of Ad4-Env recombinant following boost immunizations with soluble trimers. Rabbits were immunized with Ad4 FDE3 Envl50 or Ad4 FDE3 Envl45-NFL-TD-CD5 on weeks 0 and 4. Some rabbits were boosted with the BG505 DS-SOSIP soluble trimer on week 12 and the 16055 Del Gly4 soluble trimer on week 20. Sera was collected at weeks 12, 20, and 28. Serum neutralization was tested by a pseudovirus entry inhibition assay against 10 strains. Each row represents 1 rabbit.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file, created on July 27, 2021, 164 KB, which is incorporated by reference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleotide sequence of Ad4-Envl45NFL.

SEQ ID NO: 2 is the nucleotide sequence of the Env coding region in Ad4-Envl45NFL.

SEQ ID NO: 3 is the amino acid sequence of the Env protein encoded by Ad4-

Envl45NFL. SEQ ID NO: 4 is the nucleotide sequence of Ad4-Envl50KN.

SEQ ID NO: 5 is the nucleotide sequence of the Env coding region in Ad4-Envl50KN.

SEQ ID NO: 6 is the amino acid sequence of the Env protein encoded by Ad4-Envl50KN.

SEQ ID NO: 7 is a cleavage incompetent gpl20 amino acid sequence (AKERVVEREKE).

SEQ ID NO: 8 is a cleavage competent gpl20 amino acid sequence (AKRRVVEREKR).

SEQ ID NO: 9 is an enhanced cleavage site gpl20 amino acid sequence (RRRRRR).

SEQ ID NO: 10 is an amino acid sequence of a peptide linker (G4SK).

SEQ ID NO: 11 is an amino acid sequence of a peptide linker (G2SG2SG3S)

SEQ ID NO: 12 is the amino acid sequence of a furin cleavage site (REKR).

SEQ ID NO: 13 is the amino acid sequence of a native flexible linker (G4SG4S).

SEQ ID NO: 14 is the amino acid sequence of a single chain linker (G3SG4SG2).

SEQ ID NO: 15 is the nucleotide sequence of a forward PCR primer (AGCTCTTCACTGGGTTTGCGAC).

SEQ ID NO: 16 is the nucleotide sequence of a reverse PCR primer (TTC AGATCCCGTGG ATCTGG) .

SEQ ID NO: 17 is the nucleotide sequence encoding the HIV-1 1086C gpl50 MutC Env protein.

SEQ ID NO: 18 is the amino acid sequence of the HIV-1 1086C gpl50 MutC Env protein.

SEQ ID NO: 19 is the amino acid sequence of the HIV-1 HXB2 (clade B) Env protein.

SEQ ID NO: 20: is the amino acid sequence of the HIV-1 1086C Env protein.

DETAILED DESCRIPTION

I. Abbreviations

Ad adenovirus

AIDS acquired immunodeficiency syndrome bnAb broadly neutralizing antibody

CMV cytomegalovirus

DS disulfide bond

ELISA enzyme-linked immunosorbent assay

EM electron microscopy

Env envelope

FDE3 fully deleted E3 region gp glycoprotein

HIV human immunodeficiency virus MFI median fluorescence intensity

MPER membrane proximal external region

NFL native flexible linker

PDE3 partially deleted E3 region

SIV simian immunodeficiency virus

TM transmembrane

II. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes single or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

To facilitate review of the various embodiments, the following explanations of terms are provided:

Adenovirus: A non-enveloped virus with a liner, double-stranded DNA genome and an icosahedral capsid. There are at least 68 known serotypes of human adenovirus, which are divided into seven species (species A, B, C, D, E, F and G). Different serotypes of adenovirus are associated with different types of disease, with some serotypes causing respiratory disease (primarily species B and C), conjunctivitis (species B and D) and/or gastroenteritis (species F and G). Adenovirus type 4 (Ad4) is a species E virus that can cause acute respiratory disease and ocular disease. Adenovirus-based vectors are commonly used for a variety of therapeutic applications, including vaccine and gene therapy vectors. In some embodiments herein, the adenovirus vector is a human replication-competent Ad4 with a complete or partial deletion in the E3 region.

Adjuvant: A component of an immunogenic composition used to enhance antigenicity. In some embodiments, an adjuvant can include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). In some embodiments, the adjuvant used in a disclosed immunogenic composition is a combination of lecithin and carbomer homopolymer (such as the ADJUPEEX™ adjuvant available from Advanced BioAdjuvants, EEC; see also Wegmann, Clin Vaccine Immunol 22(9): 1004-1012, 2015). Additional adjuvants for use in the disclosed immunogenic compositions include the QS21 purified plant extract, Matrix M, AS01, MF59, and ALFQ adjuvants. Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM- CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL and toll-like receptor (TLR) agonists, such as TLR-9 agonists. The person of ordinary skill in the art is familiar with adjuvants (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed immunogens.

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid. In some examples, an amino acid in a polypeptide is substituted with an amino acid from a homologous polypeptide, for example, an amino acid in a recombinant Clade C HIV-1 Env polypeptide can be substituted with the corresponding amino acid from a Clade B HIV- 1 Env polypeptide.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects. The term “primate” includes human and non-human primates, such as macaques and rhesus monkeys. Thus, a primate includes a monkey, baboon, chimpanzee, gorilla, and a human. Nonhuman primates are appreciated to themselves be susceptible to infection by retroviruses and in particular immunodeficiency viruses and represent well-established animal models as to human response with an appreciation that physiological differences often require different doses in milligrams per kilogram for a nonhuman primate animal model relative to a human.

Antibody: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen), such as HIV-1 Env. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non- limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; singlechain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2^nd Ed., Springer Press, 2010). Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs.” The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well- known numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877 , 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik <?/ <://. (see Kunik et al. , PLoS Comput Biol 8:el002388, 2012; and Kunik et al. , Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to an epitope of an antigen.

CD4: A T-cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV-1 on T-cells during HIV-1 infection. CD5: A protein that is primarily expressed on the surface of T cells. In some embodiments herein, a modified Env protein includes a CD5 signal sequence at the N-terminus having an amino acid sequence set forth as residues 1-24 of SEQ ID NO: 3.

Cleavage competent: In the context of HIV-1, a “cleavage competent” Env is an Env with an amino acid sequence located between gpl20 and gp41 that can be cleaved, such as by furin. In some examples herein, the cleavage competent amino acid sequence comprises residues 487-497 of SEQ ID NO: 6.

Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the function of a protein, such as HIV-1 Env. “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to elicit an immune response when administered to a subject. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Furthermore, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity or function of a protein, such as a recombinant Env protein, such as the ability to elicit an immune response when administered to a subject. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

Effective amount: The amount of an agent, such as an immunogen or immunogenic composition disclosed herein (e.g., a recombinant Ad4 expressing modified HIV-1 Env), that is sufficient to elicit a desired response, such as an immune response in a subject. It is understood that to obtain a protective immune response against an antigen of interest, multiple administrations of a disclosed immunogen/immunogenic composition can be required, and/or administration of a disclosed composition as the “prime” in a prime boost protocol wherein the boost immunogen can be different from the prime immunogenic composition. Accordingly, an effective amount of a disclosed immunogen/immunogenic composition can be the amount of the immunogen or immunogenic composition sufficient to elicit a priming immune response in a subject that can be subsequently boosted with the same or a different immunogen to elicit a protective immune response.

In one example, a desired response is to elicit an immune response that inhibits or prevents HIV-1 infection. The HIV-1 infected cells do not need to be completely eliminated or prevented for the composition to be effective. For example, administration of an effective amount of an immunogen or immunogenic composition can elicit an immune response that decreases the number of HIV-1 infected cells (or prevents the infection of cells) by a desired amount, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable HIV-1 infected cells), as compared to the number of HIV-1 infected cells in the absence of the immunization.

Heterologous: Originating from a separate genetic source or species. For example, a heterologous polypeptide or polynucleotide refers to a polypeptide or polynucleotide derived from a different source or species.

Human immunodeficiency virus type 1 (HIV-1): A retrovirus that causes immunosuppression in humans (HIV-1 disease), and leads to a disease complex known as the acquired immunodeficiency syndrome (AIDS). “HIV-1 disease” refers to a well -recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by a human immunodeficiency virus, as determined by antibody or Western blot studies. Laboratory findings associated with this disease include a progressive decline in T cells. Related viruses that are used as animal models include simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Treatment of HIV- 1 with HAART (highly active antiretroviral therapy) has been effective in reducing the viral burden and ameliorating the effects of HIV- 1 infection in infected individuals.

HIV-1 envelope protein (Env): The HIV-1 Env protein is initially synthesized as a precursor protein of 845-870 amino acids in length. Individual precursor polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease between approximately positions 511/512 to generate separate gpl20 and gp41 polypeptide chains, which remain associated as gpl20-gp41 protomers within the homotrimer. The ectodomain (that is, the extracellular portion) of the HIV-1 Env trimer undergoes several structural rearrangements from a prefusion closed conformation that evades antibody recognition, through intermediate conformations that bind to receptor CD4 and coreceptor (either CCR5 or CXCR4), to a postfusion conformation. The HIV-1 Env ectodomain comprises the gpl20 protein (approximately HIV-1 Env positions 31-511) and the gp41 ectodomain (approximately HIV-1 Env positions 512-664). An HIV-1 Env ectodomain trimer comprises a protein complex of three HIV-1 Env ectodomains.

Mature gpl20 includes approximately HIV-1 Env residues 31-511, contains most of the external, surface-exposed domains of the HIV-1 Env trimer, and binds to both cellular CD4 receptors and cellular chemokine receptors (such as CCR5). The mature gpl20 wild-type polypeptide is heavily N-glycosylated, giving rise to an apparent molecular weight of 120 kD. Native gpl20 includes five conserved regions (C1-C5) and five regions of high variability (V1-V5).

Mature gp41 includes approximately HIV-1 Env residues 512-860, and possesses a cytosolic domain, a transmembrane domain, and an ectodomain. The gp41 ectodomain (including approximately HIV-1 Env residues 512-644) can interact with gpl20 to form an HIV-1 Env protomer that trimerizes to form the HIV-1 Env trimer.

A standardized numbering scheme for HIV-1 proteins (the HXB2 numbering scheme) is set forth in Numbering Positions in HIV Relative to HXB2CG, Bette Korber et al., Human Retroviruses and AIDS 1998: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Korber et al., Eds. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM, which is incorporated by reference herein in its entirety. For reference, the amino acid sequence of HIV-1 Env of HXB2 (clade B) is set forth as SEQ ID NO: 19 (GENBANK® GI: 1906382, incorporated by reference herein).

HXB2 Env (SEQ ID NO: 19):

MRVKEKYQHLWRWGWRWGTMLLGMLMICSATEKLWVTVYYGVPVWKEATTTLFCAS DAKAYDTEVHNVWATHACVPTDPNPQEVVLVNVTENFNMWKNDMVEQMHEDIISLWD QSLKPCVKLTPLCVSLKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAF FYKLDIIPIDNDTTSYKLTSCNTSVITQACPKVSFEPIPIHYCAPAGFAILKCNNKTFNGTGPC TNVSTVQCTHGIRPVVSTQLLLNGSLAEEEVVIRSVNFTDNAKTIIVQLNTSVEINCTRPNN NTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIASKLREQFGNNKTIIFKQ SSGGDPEIVTHSFNCGGEFFYCNSTQLFNSTWFNSTWSTEGSNNTEGSDTITLPCRIKQIINM WQKVGKAMYAPPISGQIRCSSNITGLLLTRDGGNSNNESEIFRPGGGDMRDNWRSELYKY KVVKIEPLGVAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSTMGAASMTLTVQARQLLS GIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTA VPWNASWSNKSLEQIWNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKW ASLWNWFNITNWLWYIKLFIMIVGGLVGLRIVFAVLSIVNRVRQGYSPLSFQTHLPTPRGP DRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRG

WEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGACRAIRHIPRRIRQ GLERILL

The modified Env polypeptides disclosed herein are derived from clade C, strain 1086 (1086C). Thus, a “modified” Env polypeptide is an Env that is modified with respect to the sequence of the WT 1086C Env protein. The amino acid sequence of the WT 1086C Env is set forth herein as SEQ ID NO: 20.

1086C Env (SEQ ID NO: 20):

MRVRGIWKNWPQWLIWSILGFWIGNMEGSWVTVYYGVPVWKEAKTTLFCASDAKAYEK EVHNVWATHACVPTDPNPQEMVLANVTENFNMWKNDMVEQMHEDIISLWDESLKPCVK LTPLCVTLNCTNVKGNESDTSEVMKNCSFKATTELKDKKHKVHALFYKLDVVPLNGNSSS SGEYRLINCNTSAITQACPKVSFDPIPLHYCAPAGFAILKCNNKTFNGTGPCRNVSTVQCTH GIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVNIVCTRPNNNTRKSIRIGPGQT FYATGDIIGNIRQAHCNINESKWNNTLQKVGEELAKHFPSKTIKFEPSSGGDLEITTHSFNCR GEFFYCNTSDLFNGTYRNGTYNHTGRSSNGTITLQCKIKQIINMWQEVGRAIYAPPIEGEIT CNSNITGLLLLRDGGQSNETNDTETFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTEAK RRVVEREKRAVGIGAVFLGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQSNLLRAIEAQ QHMLQLTVWGIKQLQARVLAIERYLKDQQLLGMWGCSGKLICTTAVPWNSSWSNKSQNE IWGNMTWMQWDREINNYTNTIYRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDISKWL

WYIKIFIMIIGGLIGLRIIFAVLSIVKRVRQGYSPLSFQTLTPNPRGPDRPGGIEEEGGEQDRDR SARLASGFLTLVWDDLRSLCLFSYHLLRDFILVATRTVELLGRSSLKGLQRGWEILRYLGSL VQYWGLELKKSAISLIDTIAIAVAERTDRIIEIVQRICRAIRNIPRRIRQGFETALL

HIV-1 Envl45: A recombinant HIV Env polypeptide including gpl20, the gp41 ectodomain, and the gp41 transmembrane domain, but not the gp41 cytoplasmic domain (see FIG. 1A and FIG. 2).

HIV-1 Envl50: A recombinant HIV Env polypeptide including gpl20, the gp41 ectodomain, the gp41 transmembrane domain and a truncated gp41 cytosolic domain (see FIG. 1A and FIG. 2).

HIV-1 gpl60: A recombinant HIV Env polypeptide including gpl20 and the entire gp41 protein (ectodomain, transmembrane domain, and cytosolic tail; see FIG. 1A).

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some embodiments, the response is specific for a particular antigen (an “antigen- specific response”), such as an HIV-1 Env. In some embodiments, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In other embodiments, the response is a B cell response, and results in the production of specific antibodies. “Priming an immune response” refers to treatment of a subject with a “prime” immunogen/immunogenic composition to induce an immune response that is subsequently “boosted” with a boost immunogen/immunogenic composition. Together, the prime and boost immunizations produce the desired immune response in the subject. “Enhancing an immune response” refers to coadministration of an adjuvant and an immunogenic agent, wherein the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to the subject in the absence of the adjuvant.

Immunogen: A protein or a portion thereof that is capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen (e.g. HIV). In some embodiments, the immunogen is HIV-1 Env, such as Env expressed by a recombinant Ad4.

Immunogenic composition: A composition that includes an immunogen or a nucleic acid molecule or vector encoding an immunogen (such as HIV-1 Env), that elicits a measurable CTL response against the immunogen, and/or elicits a measurable B cell response (such as production of antibodies) against the immunogen, when administered to a subject. It further refers to isolated nucleic acids encoding an immunogen, such as a nucleic acid that can be used to express the immunogen (and thus be used to elicit an immune response against this immunogen). For in vivo use, the immunogenic composition can include the protein or nucleic acid molecule in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as acquired immunodeficiency syndrome (AIDS). “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated.

Linker: One or more peptides positioned between two moieties. In several embodiments, a peptide linker can be used to link the C-terminus of a first protein to the N-terminus of a second protein. Non- limiting examples of peptide linkers include glycine-serine peptide linkers, such as the linkers set forth herein as SEQ ID NOs: 10, 11, 13 and 14. Such linkage can be accomplished, for example, using molecular biology techniques to genetically manipulate DNA encoding the first polypeptide linked to the second polypeptide by the peptide linker.

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (including rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens and immunogenic compositions.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to elicit the desired anti-HIV-1 immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

Plurality of amino acid substitutions: At least two amino acid substitutions, including at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 amino acid substitutions.

Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues. Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by the artificial manipulation of isolated segments of nucleic acids, for example, using genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several embodiments, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

Sequence identity: The similarity between amino acid or nucleotide sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide or polynucleotide will possess a relatively high degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are known. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; and Pearson et al. , Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.

Variants of a polypeptide or nucleic acid sequence are typically characterized by possession of at least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full length alignment with the amino acid or nucleotide sequence of interest. Sequences with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids (or 30-60 nucleotides), and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet.

As used herein, reference to “at least 90% identity” (or similar language) refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

Signal Peptide: A short amino acid sequence (e.g., approximately 18-30 amino acids in length) that directs newly synthesized secretory or membrane proteins to and through membranes (for example, the endoplasmic reticulum membrane). Signal peptides are typically located at the N-terminus of a polypeptide and are removed by signal peptidases after the polypeptide has crossed the membrane. Signal peptide sequences typically contain three common structural features: an N- terminal polar basic region (n-region), a hydrophobic core, and a hydrophilic c-region). In some embodiments herein, the signal peptide is a CD5 signal sequence set forth as residues 1-24 of SEQ ID NO: 3. In other embodiments, the signal peptide is a native Env signal sequence set forth as residues 1-25 of SEQ ID NO: 6.

Subject: Living multicellular vertebrate organisms, a category that includes human and non-human mammals. In some embodiments, the subject is a human. In some examples, a subject who is in need of inhibiting or preventing an HIV-1 infection is selected. For example, the subject can be uninfected and at risk of HIV-1 infection.

Therapeutically effective amount and prophylactically effective amount: A quantity of a specific substance, such as a disclosed immunogen or immunogenic composition, sufficient to achieve a desired effect in a subject being treated, such as a protective immune response. A “therapeutically effective amount” can be the amount necessary to inhibit HIV replication or treat AIDS in a subject with an existing HIV infection. “Prophylactically effective amount” refers to administration of an agent or composition that inhibits or prevents establishment of an infection, such infection by HIV. Pre-exposure prophylaxis (PrEP) is the prevention or inhibition of an HIV infection in a host, wherein the active agent(s) are administered prior to any possible infection (e.g., prior to any exposure) of the subject with the virus. As noted above, a “protective” treatment for HIV inhibits infection of the subject when the subject is subsequently exposed to the virus. “Protection” as used in the context of a host response to HIV challenge results in the host being serologically negative and negative in a polymerase chain reaction (PCR) testing for viral genome.

Vaccine: A pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen- specific immune response to an antigen of a pathogen, for example a viral pathogen, or to a cellular constituent correlated with a pathological condition. A vaccine may include a polynucleotide (such as a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (such as a disclosed antigen), a virus, a cell or one or more cellular constituents. In one specific, non-limiting example, a vaccine reduces the severity of the symptoms associated with HIV-1 infection and/or decreases the viral load compared to a control. In another non-limiting example, a vaccine reduces HIV-1 infection compared to a control.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s) that is operationally linked to the coding sequence of a protein (such as an immunogenic protein) of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. Non-limiting examples of viral vectors include adenovirus vectors, adeno- associated virus (AAV) vectors, and poxvirus vectors (e.g., vaccinia, fowlpox).

VRC01: A broadly neutralizing monoclonal antibody that specifically binds to the CD4 binding site on HIV-1 Env and can inhibit HIV-1 infection of target cells. The person of ordinary skill in the art is familiar with the VRC01 mAb and with methods of its use and production (see, for example, Wu et al., Science, 329(5993):856-861, 2010, and PCT Publication No. WO 2012/154312, each of which is incorporated by reference herein). The amino acid sequences of the heavy and light variable regions of the VRC01 mAb are known and have been deposited in GenBank® as Nos. ADF47181.1 (VRC01 V_H) and ADF47184.1 (VRC01 V_L), each of which is incorporated by reference herein).

Unit dosage form: A physically discrete unit, such as a capsule, tablet, or solution, that is suitable as a unitary dosage for a human patient, each unit containing a predetermined quantity of one or more active ingredient(s) calculated to produce a therapeutic effect, in association with at least one pharmaceutically acceptable diluent or carrier, or combination thereof. Unit dosage formulations contain a daily dose or an appropriate fraction thereof, of the active ingredient(s).

Virus: Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. “Viral replication” is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the “immunodeficiency viruses” which include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV).

III. Modified HIV-1 Envelope (Env) Polypeptides

The present disclosure characterizes various HIV Env designs to identify recombinant Env proteins having a native-like conformation. An extensive array of modified Env proteins based on HIV-1 clade C, strain 1086 (“1086C”) was generated and the impact of Env design changes on antigenicity and immunogenicity was examined (see Examples 1 and 2). Based on these studies, two modified Env polypeptides (Envl45NFL and Envl50KN) were selected for further characterization. Nucleic acid sequences encoding Envl45NFL and Envl50KN were cloned into a replication competent Ad4 vector to produce Ad4-Envl45NFL (also referred to as “Ad4-Envl45- NFL-TD-CD5”) and Ad4-Envl50KN, respectively. Ad4-Envl45NFL expresses stabilized and homogeneous Env polypeptides. Ad4-Envl50KN expresses non- stabilized and less uniform Env polypeptides (see FIG. 8).

A. Envl45NFL (Stabilized Env)

Amino acid sequence of Envl45NFL (SEQ ID NO: 3) MPMGSLQPLATLYLLGMLVASVLAMEGSWVTVYYGVPVWKDAETTLFCASDAKAYE KEKHNVWATHACVPTDPNPQEMVLANVTENFNMWKNDMVEQMHTDIISLWDESLKPCV KLNPLCVTLNCTNVKGNESDTSEVMKNCSFNATTELRDKKQKVHALFYKLDVVPLNGNS SSSGEYRLINCNTSAITQACPKVSFDPIPLHYCAPAGFAILKCNNKTFNGTGPCRNVSTVQC THGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVNIVCTRPNNYTRKSIRIGPG QTFYAMGDIIGNIRQAHCNINESKWNNTLQKVGEELAKHFPSKTIKFEPSSGGDLEITTHSF NCRGEFFYCNTSDLFNGTYRNGTYNHTGRSSNGTITLQCKIKQIINMWQRVGQAIYAPPIEG EITCNSNITGLLLLRDGGQSNETNDTETFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTR AKRRVVEGGGGSGGGGSAYGIGAVRRGFLGAAGSTMGAASMTLTVQARQLLSGIVQQQS NLLRAPEAQQHMLQGTVWGIKQLQARVLAIERYLKDQQLLGMWGCSGKLICTTAVPWNS SWSNKSQNEIWGNMTWMQWDREIGNYTNTIYRLLEDSQNQQEKNEKDLLALDSWKNLW NWFDISKWLWYIKGGGGSAIFIMIIGGLIGLRIIFAVLSI

Envl45NFL includes the CD5 signal sequence (residues 1-24 of SEQ ID NO: 3; shown in bold underline above), 18 amino acid substitutions (indicated by underline) relative to WT 1086C Env (set forth herein as SEQ ID NO: 20), a native flexible linker (NFL) at the C-terminus of gpl20 (residues 493-502 of SEQ ID NO: 3; shown in bold italics), a G4SK linker (SEQ ID NO: 10) preceding the transmembrane domain of gp41 (residues 675-680 of SEQ ID NO: 3; shown in bold italics), and a C-terminal truncation of gp41, which deletes the cytoplasmic domain. The C- terminal truncation increases cell surface expression of Env.

Table 1 provides a list of the 18 amino acid substitutions present in Envl45NFL, relative to WT 1086C Env. The residue numbers of each substitution are based on the HXB2 numbering scheme. The amino acid position in SEQ ID NO: 3 that corresponds to each substitution is also provided.

Table 1. Amino acid substitutions relative to WT 1086C Env (SEQ ID NO: 20)

B. Envl50KN (Non-Stabilized Env)

Amino acid sequence of Envl50KN (SEQ ID NO: 6)

MRVRGIWKNWPQWLIWSILGFWIGNMEGSWVTVYYGVPVWKEAKTTLFCASDAKAY EKEVHNVWATHACVPTDPNPQEMVLANVTENFNMWKNDMVEQMHEDIISLWDESLKPC VKLTPLCVTLNCTNVKGNESDTSEVMKNCSFNATTELKDKKHKVHALFYKLDVVPLNGN SSSSGEYRLINCNTSAITQACPKVSFDPIPLHYCAPAGFAILKCNNKTFNGTGPCRNVSTVQC THGIKPVVSTQLLLNGSLAEEEIIIRSENLTNNAKTIIVHLNESVNIVCTRPNNNTRKSIRIGPG QTFYATGDIIGNIRQAHCNINESKWNNTLQKVGEELAKHFPSKTIKFEPSSGGDLEITTHSFN CRGEFFYCNTSDLFNGTYRNGTYNHTGRSSNGTITLQCKIKQIINMWQEVGRAIYAPPIEGE ITCNSNITGLLLLRDGGQSNETNDTETFRPGGGDMRDNWRSELYKYKVVEIKPLGVAPTEA KRR WEREKR A VGIGA VFEGFEG AAGSTMGAASMTETVQARQEES GIVQQQSNLLRAIEA QQHMLQLTVWGIKQLQARVLAIERYLKDQQLLGMWGCSGKLICTTAVPWNSSWSNKSQ

NEIWGNMTWMQWDREINNYTNTIYRLLEDSQNQQEKNEKDLLALDSWKNLWNWFDISK WLWYIKIFIMIIGGLIGLRIIFAVLSIVKRVRQGYSPLSFQTLTPNPRGPDRPGGIEEEGGEQD RDRS

Envl50KN includes the native gpl20 signal sequence (residues 1-25 of SEQ ID NO: 6; shown in bold underline above), the K160N mutation (at position 146 of SEQ ID NO: 6; underlined), the native furin cleavage site at the C-terminus of gpl20 (residues 487-497 of SEQ ID NO: 6; shown in bold italics) and a C-terminal truncation of gp41, which deletes a portion of the cytosolic domain. The C-terminal truncation increases cell surface expression of Env.

C. Env polypeptides and Env-expressing recombinant adenoviruses

Disclosed herein are modified HIV-1 Env polypeptides. The Env polypeptides are based on HIV-1 clade C strain 1086 (“1086C”).

In some embodiments, the modified Env polypeptide includes a heterologous CD5 signal peptide sequence at the N-terminus; a plurality of amino acid substitutions to stabilize Env trimer formation; an asparagine substitution at position 160 (numbered with reference to the HXB2 numbering scheme); a proline substitution at position 559 (numbered with reference to the HXB2 numbering scheme); a first heterologous peptide linker positioned between the gpl20 and gp41 subunits; a second heterologous peptide linker positioned between the ectodomain and the transmembrane domain; and a C-terminal truncation that results in deletion of the cytoplasmic domain. In other embodiments, the Env polypeptide does not include a signal sequence. In some examples, the CD5 signal peptide sequence includes residues 1-24 of SEQ ID NO:

3.

In some examples, the plurality of amino acid substitutions includes an aspartic acid substitution at position 47; a glutamic acid substitution at position 49; a lysine substitution at position 65; a threonine substitution at position 106; an arginine substitution at position 166; a glutamine substitution at position 170; a tyrosine substitution at position 302; a methionine substitution at position 320; an arginine substitution at position 429; a glutamine substitution at position 432; an arginine substitution at position 500; a tyrosine substitution at position 513; an arginine substitution at position 519; an arginine substitution at position 520; a glycine substitution at position 568; a glycine substitution at position 636; or any combination thereof, wherein the amino acid position is numbered with reference to the HXB2 numbering scheme. In particular examples, the modified Env polypeptide includes two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more of the recited substitutions. In specific examples, the modified Env polypeptide includes all 16 substitutions.

In some examples, the first heterologous peptide linker includes the amino acid sequence G4SG4S (SEQ ID NO: 13).

In some examples, the second heterologous peptide linker includes the amino acid sequence G₄SK (SEQ ID NO: 10).

In some examples, the C-terminal truncation results in an Env 145 polypeptide.

In some examples, the amino acid sequence of the Env polypeptide is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3. In specific non-limiting examples, the amino acid sequence of the Env polypeptide comprises or consists of SEQ ID NO: 3.

In other embodiments, the modified Env polypeptide includes a native HIV-1 signal peptide at the N-terminus; an asparagine substitution at position 160, numbered with reference to the HXB2 numbering scheme; a cleavage competent sequence positioned between the gpl20 and gp41 subunits; and a C-terminal truncation that results in deletion of a portion of the cytoplasmic domain.

In some examples, the cleavage competent sequence includes residues 487-497 of SEQ ID NO: 6.

In some examples, the C-terminal truncation results in an Envl50 polypeptide.

In some examples, the amino acid sequence of the Env polypeptide is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6. In specific non-limiting examples, the amino acid sequence of the Env polypeptide comprises or consists of SEQ ID NO: 6.

Env trimers that include the modified Env polypeptides disclosed herein are further provided.

Also provided are recombinant adenoviruses that express a modified 1086C Env polypeptide disclosed herein. In some embodiments, the adenovirus is a replication competent adenovirus. In specific examples, the adenovirus is a replication competent Ad4.

In some embodiments, the genome of the recombinant adenovirus includes a complete or partial deletion of the E3 region.

In some embodiments, the genome of the recombinant adenovirus is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1. In some examples, the genome of the recombinant adenovirus comprises or consists of SEQ ID NO: 1.

In some embodiments, the genome of the recombinant adenovirus is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4. In some examples, the genome of the recombinant adenovirus comprises or consists of SEQ ID NO: 4.

IV. Polynucleotides and Expression

Polynucleotides encoding a disclosed immunogen (e.g., a modified Env polypeptide) are also provided. These polynucleotides include DNA, cDNA and RNA sequences that encode the antigen. One of skill in the art can readily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids which differ in sequence but which encode the same protein sequence, or encode a conjugate or fusion protein including the nucleic acid sequence.

In some embodiments, the modified Env polypeptide is encoded by the nucleic acid sequence set forth as SEQ ID NO: 2 (Envl45NFL): ATGCCCATGGGGTCTCTGCAACCGCTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGC TTCCGTGCTAGCTATGGAGGGCTCCTGGGTCACCGTGTATTACGGCGTGCCCGTGTGGA AGGATGCCGAGACAACACTGTTCTGTGCCAGCGACGCCAAGGCCTACGAGAAAGAAA AGCACAACGTGTGGGCCACTCACGCCTGCGTGCCAACCGATCCTAATCCTCAAGAGAT GGTGCTGGCCAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTCGAGCA GATGCACACCGACATCATCAGCCTGTGGGACGAGAGCCTGAAGCCTTGCGTGAAGCTG AACCCTCTGTGCGTGACCCTGAACTGCACCAACGTGAAGGGCAACGAGAGCGACACC AGCGAAGTGATGAAGAACTGCAGCTTCAACGCCACCACCGAGCTGCGGGACAAGAAA

CAGAAAGTGCACGCCCTGTTCTACAAGCTGGACGTGGTGCCCCTGAACGGCAATAGCT

CTAGCAGCGGCGAGTACCGGCTGATCAACTGCAACACCAGCGCCATCACTCAGGCCTG

TCCTAAGGTGTCCTTCGATCCCATTCCTCTGCACTACTGTGCCCCTGCCGGATTCGCCA

TCCTGAAGTGCAACAACAAGACCTTCAACGGCACAGGCCCCTGCAGAAACGTGTCCAC

CGTGCAGTGTACCCACGGCATCAAGCCAGTGGTGTCTACCCAGCTGCTGCTGAATGGC

TCTCTGGCCGAGGAAGAGATCATCATCAGAAGCGAGAACCTGACCAACAACGCCAAG

ACCATCATCGTGCACCTGAACGAGTCCGTGAACATCGTGTGCACCCGGCCTAACAACT

ACACCAGAAAGAGCATCCGGATCGGCCCTGGCCAGACCTTTTATGCCATGGGCGATAT

CATCGGCAATATCAGACAGGCCCACTGTAACATCAACGAGTCCAAGTGGAACAACACC

CTCCAGAAAGTGGGCGAAGAACTCGCCAAGCACTTCCCCAGCAAGACAATCAAGTTCG

AGCCCAGCTCTGGCGGCGACCTGGAAATCACCACACACAGCTTCAATTGCCGGGGAGA

GTTCTTCTACTGCAATACCTCCGACCTGTTCAATGGCACCTACCGGAACGGCACCTACA

ACCACACAGGCAGAAGCAGCAACGGCACCATCACTCTGCAGTGCAAGATCAAGCAGA

TCATCAATATGTGGCAGAGAGTCGGCCAGGCCATCTACGCCCCTCCAATTGAGGGCGA

GATCACCTGTAACAGCAACATCACCGGCCTGCTGCTGCTCAGAGATGGCGGCCAGAGC

AACGAGACAAACGACACCGAGACATTCAGACCCGGCGGAGGCGACATGAGAGACAAT

TGGAGAAGCGAGCTGTACAAGTACAAGGTGGTGGAAATCAAGCCCCTGGGCGTCGCC

CCTACCAGAGCTAAGAGAAGAGTGGTTGAAGGCGGCGGAGGAAGCGGAGGCGGAGG

ATCTGCTTATGGAATCGGCGCCGTGCGGAGAGGCTTTCTTGGAGCTGCTGGCTCTACA

ATGGGCGCTGCCAGCATGACACTGACCGTGCAGGCTAGACAGCTGCTGAGCGGAATTG

TCCAGCAGCAGAGCAACCTGCTGAGAGCCCCTGAAGCACAGCAGCACATGCTGCAGG

GAACAGTGTGGGGAATCAAACAGCTGCAGGCCAGAGTGCTGGCTATCGAGAGATACC

TGAAGGACCAGCAACTGCTCGGCATGTGGGGCTGTTCTGGCAAGCTGATCTGTACCAC

AGCCGTGCCTTGGAACAGCTCCTGGTCCAACAAGAGCCAGAACGAGATCTGGGGCAA

CATGACCTGGATGCAGTGGGACAGAGAGATCGGCAATTACACCAACACCATCTACCGG

CTGCTGGAAGATAGCCAGAACCAGCAAGAGAAGAACGAGAAGGACCTGCTGGCCCTG

GACAGCTGGAAAAACCTGTGGAATTGGTTCGACATCAGCAAGTGGCTCTGGTACATCA

AAGGTGGCGGCGGAAGCAAGATCTTCATCATGATCATCGGCGGCCTGATCGGCCTGCG GATCATTTTTGCCGTGCTGAGCATCTGA

In other embodiments, the modified Env polypeptide is encoded by the nucleic acid sequence set forth as SEQ ID NO: 5 (Envl50KN): ATGCGCGTGCGCGGCATCTGGAAGAACTGGCCCCAGTGGCTGATCTGGTCCATCCTGG

GCTTCTGGATCGGCAACATGGAGGGCTCCTGGGTGACCGTGTACTACGGCGTGCCCGT

GTGGAAGGAGGCCAAGACCACCCTGTTCTGCGCCTCCGACGCCAAGGCCTACGAGAA

GGAGGTGCACAACGTGTGGGCCACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAG

GAGATGGTGCTGGCCAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTG

GAGCAGATGCACGAGGACATCATCTCCCTGTGGGACGAGTCCCTGAAGCCCTGCGTGA

AGCTGACCCCCCTGTGCGTGACCCTGAACTGCACCAACGTGAAGGGCAACGAGTCCGA

CACCTCCGAGGTGATGAAGAACTGCTCCTTCAACGCCACCACCGAGCTGAAGGACAAG

AAGCACAAGGTGCACGCCCTGTTCTACAAGCTGGACGTGGTGCCCCTGAACGGCAACT

CCTCCTCCTCCGGCGAGTACCGCCTGATCAACTGCAACACCTCCGCCATCACCCAGGCC

TGCCCCAAGGTGTCCTTCGACCCCATCCCCCTGCACTACTGCGCCCCCGCCGGCTTCGC

CATCCTGAAGTGCAACAACAAGACCTTCAACGGCACCGGCCCCTGCCGCAACGTGTCC

ACCGTGCAGTGCACCCACGGCATCAAGCCCGTGGTGTCCACCCAGCTGCTGCTGAACG

GCTCCCTGGCCGAGGAGGAGATCATCATCCGCTCCGAGAACCTGACCAACAACGCCAA

GACCATCATCGTGCACCTGAACGAGTCCGTGAACATCGTGTGCACCCGCCCCAACAAC

AACACCCGCAAGTCCATCCGCATCGGCCCCGGCCAGACCTTCTACGCCACCGGCGACA

TCATCGGCAACATCCGCCAGGCCCACTGCAACATCAACGAGTCCAAGTGGAACAACAC

CCTGCAGAAGGTGGGCGAGGAGCTGGCCAAGCACTTCCCCTCCAAGACCATCAAGTTC

GAGCCCTCCTCCGGCGGCGACCTGGAGATCACCACCCACTCCTTCAACTGCCGCGGCG

AGTTCTTCTACTGCAACACCTCCGACCTGTTCAACGGCACCTACCGCAACGGCACCTAC

AACCACACCGGCCGCTCCTCCAACGGCACCATCACCCTGCAGTGCAAGATCAAGCAGA

TCATCAACATGTGGCAGGAGGTGGGCCGCGCCATCTACGCCCCCCCCATCGAGGGCGA

GATCACCTGCAACTCCAACATCACCGGCCTGCTGCTGCTGCGCGACGGCGGCCAGTCC

AACGAGACCAACGACACCGAGACCTTCCGCCCCGGCGGCGGCGACATGCGCGACAAC

TGGCGCTCCGAGCTGTACAAGTACAAGGTGGTGGAGATCAAGCCCCTGGGCGTGGCCC

CCACCGAGGCCAAGCGCCGCGTGGTGGAGCGCGAGAAGCGCGCCGTGGGCATCGGCG

CCGTGTTCCTGGGCTTCCTGGGCGCCGCCGGCTCCACCATGGGCGCCGCCTCCATGACC

CTGACCGTGCAGGCCCGCCAGCTGCTGTCCGGCATCGTGCAGCAGCAGTCCAACCTGC

TGCGCGCCATCGAGGCCCAGCAGCACATGCTGCAGCTGACCGTGTGGGGCATCAAGCA

GCTGCAGGCCCGCGTGCTGGCCATCGAGCGCTACCTGAAGGACCAGCAGCTGCTGGGC

ATGTGGGGCTGCTCCGGCAAGCTGATCTGCACCACCGCCGTGCCCTGGAACTCCTCCT

GGTCCAACAAGTCCCAGAACGAGATCTGGGGCAACATGACCTGGATGCAGTGGGACC

GCGAGATCAACAACTACACCAACACCATCTACCGCCTGCTGGAGGACTCCCAGAACCA

GCAGGAGAAGAACGAGAAGGACCTGCTGGCCCTGGACTCCTGGAAGAACCTGTGGAA CTGGTTCGACATCTCCAAGTGGCTGTGGTACATCAAGATCTTCATCATGATCATCGGCG GCCTGATCGGCCTGCGCATCATCTTCGCCGTGCTGAGCATCGTGAAGCGCGTGCGCCA GGGCTACAGCCCCCTGAGCTTCCAGACCCTGACCCCCAACCCCCGCGGCCCCGACCGC CCCGGCGGCATCGAGGAGGAGGGCGGCGAGCAGGACCGCGACCGCAGCTAA

Provided are nucleic acid molecules encoding a modified 1086C Env polypeptide as disclosed herein.

In some embodiments, the nucleic acid molecule includes a nucleotide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2. In some examples, the nucleotide sequence comprises or consists of SEQ ID NO: 2.

In other embodiments, the nucleic acid molecule includes a nucleotide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 5. In some examples, the nucleotide sequence comprises or consists of SEQ ID NO: 5.

Also provided are vectors that include a nucleic acid molecule encoding a modified 1086C Env polypeptide. In some embodiments, the vector is a viral vector.

The disclosed nucleic acid sequences encode a protomer of an Env polypeptide and when expressed in cells under appropriate conditions, form HIV-1 Env trimers.

Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are known (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^th ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013)). Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include, for example, polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

The polynucleotides encoding a disclosed immunogen can include a recombinant DNA which is incorporated into a vector, an autonomously replicating plasmid, a virus, or the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single and double stranded forms of DNA.

Polynucleotide sequences encoding a disclosed immunogen can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to, appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

DNA sequences encoding the disclosed immunogen can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable host cells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines. Techniques for the propagation of mammalian cells in culture are well-known (see, e.g., Helgason and Miller (Eds.), 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4^th Ed., Humana Press). Examples of commonly used mammalian host cell lines are VERO, HeLa, CHO, WI38, BHK, and COS cell lines, although alternative cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features. In some embodiments, the host cells include HEK293 cells or derivatives thereof, such as GnTI ^/_ cells (ATCC® No. CRL-3022), or HEK-293F cells. Transformation of a host cell with recombinant DNA can be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as, but not limited to, E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCh method using procedures well known in the art. Alternatively, MgCh or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or viral vectors can be used. Eukaryotic cells can also be co-transformed with polynucleotide sequences encoding a disclosed antigen, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein (see for example, Viral Expression Vectors, Springer press, Muzyczka ed., 2011). One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines.

In one non-limiting example, a disclosed immunogen is expressed using the pVRC8400 vector (described in Barouch et al., J. Virol, 79 ,8828-8834, 2005, which is incorporated by reference herein).

Modifications can be made to a nucleic acid encoding a disclosed immunogen without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, termination codons, a methionine added at the amino terminus to provide an initiation site, additional amino acids placed on either terminus to create conveniently located restriction sites, or additional amino acids (such as poly His) to aid in purification steps.

V. Adenovirus and Other Viral Vectors

A nucleic acid molecule encoding a disclosed immunogen can be included in a viral vector, for example, for expression of the immunogen in a host cell, or for immunization of a subject. In some embodiments, the viral vectors are administered to a subject as part of a prime-boost vaccination. In several embodiments, the viral vectors are included in a vaccine, such as a primer vaccine or a booster vaccine for use in a prime-boost vaccination. In several examples, the viral vector can be replication-competent. The viral vector also can be conditionally replication-competent. In other examples, the viral vector is replication-deficient in host cells.

A number of viral vectors have been constructed, that can be used to express the disclosed antigens, including polyoma, e.g., SV40 (Madzak et al., 1992, J. Gen. Virol., 73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol., 158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia et al. , 1992, J. Virol., 66:4407-4412; Quantin et al. , 1992, Proc. Natl. Acad. Sci. USA, 89:2581-2584; Rosenfeld et al. , 1992, Cell, 68:143-155; Wilkinson et al. , 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241- 256), vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene, 89:279- 282), herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al., 1992, Hum. Gene Ther. 3:11- 19; Breakfield et al. , 1987, Mo/. Neurobiol., 1:337-371; Fresse et o/., 1990, Biochem. Pharmacol., 40:2189-2199), Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol., 66:3291-3297), murine (Miller, 1992, Curr. Top. Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mo/. Cell Biol., 5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol., 64:5370-5276; Buchschalcher et al., 1992, 7. Virol., 66:2731-2739). B aculo virus (Autographa califomica multinuclear polyhedrosis virus; AcMNPV) vectors are also known in the art, and may be obtained from commercial sources (such as PharMingen, San Diego, CA; Protein Sciences Corp., Meriden, CT; Stratagene, La Jolla, CA).

In several embodiments, the viral vector can include an adenoviral vector that expresses a disclosed immunogen. Adenovirus from various origins, subtypes, or mixture of subtypes can be used as the source of the viral genome for the adenoviral vector. For example, human adenovirus can be used as the source of the viral genome for the adenoviral vector. Human adenovirus can be of various subgroups or serotypes. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36-39, and 42-48), subgroup E (e.g., serotype 4), subgroup F (e.g., serotypes 40 and 41), an unclassified serogroup (e.g., serotypes 49 and 51), or any other adenoviral serotype. In some embodiments herein, the adenovirus is human adenovirus serotype 4 (Ad4).

Non-human adenovirus (e.g., simian, chimpanzee, gorilla, avian, canine, ovine, or bovine adenoviruses) can also be used to generate the adenoviral vector. For example, a simian adenovirus can be used as the source of the viral genome of the adenoviral vector. A simian adenovirus can be of serotype 1, 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. A simian adenovirus can be referred to by using any suitable abbreviation known in the art, such as, for example, SV, SAdV, SAV or sAV. In some examples, a simian adenoviral vector is a simian adenoviral vector of serotype 3, 7, 11, 16, 18, 19, 20, 27, 33, 38, or 39. In one example, a chimpanzee serotype C Ad3 vector is used (see, e.g., Peruzzi et al., Vaccine, 27:1293-1300, 2009).

The person of ordinary skill in the art is familiar with replication competent and deficient adenoviral vectors (including singly and multiply replication deficient adenoviral vectors). Examples of replication-deficient adenoviral vectors, including multiply replication-deficient adenoviral vectors, are disclosed in U.S. Patent Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, and International Patent Application Nos. WO 94/28152, WO 95/02697, WO 95/16772, WO 95/34671, WO 96/22378, WO 97/12986, WO 97/21826, and WO 03/022311.

In some embodiments, the adenovirus vector is a replication competent adenovirus. In specific examples, the adenovirus vector is a replication competent Ad4 vector (see, e.g., Alexander et al., PLoS ONE 8(12): e82380, 2013; Alexander et al., PLoS ONE 7(2): e31177, 2012; both of which are incorporated herein by reference). The replication-competent Ad4 vector disclosed herein is based upon the orally administered U.S. military Ad4 vaccine. In some examples, the Ad4 vector includes a complete or partial deletion of the E3 region to accommodate insertion of the coding sequence for the modified Env polypeptide.

Provided herein are vectors that include a nucleic acid molecule encoding a modified 1086C Env polypeptide. In some embodiments, the vector is an adenovirus vector, such as a replication- competent adenovirus vector. In some examples, the adenovirus vector is a replication competent Ad4 vector. In particular examples, the Ad4 vector includes a deletion of the adenovirus E3 region. The deletion in E3 can be a partial deletion or a complete deletion of E3.

In some embodiments, the adenovirus vector includes a nucleotide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1. In some examples, the nucleotide sequence comprises or consists of SEQ ID NO: 1. In other embodiments, the adenovirus vector includes a nucleotide sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4. In some examples, the nucleotide sequence comprises or consists of SEQ ID NO: 4.

VI. Immunogenic Compositions

Immunogenic compositions that include a disclosed immunogen (e.g., a modified Env polypeptide, an Env trimer comprising a modified Env, or a recombinant Ad expressing a modified Env), and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to subjects by a variety of administration modes, for example, intranasal, oral, intramuscular, subcutaneous, intravenous, intra-arterial, intra- articular, intraperitoneal, or parenteral routes. Methods for preparing administrable compositions are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19^th Ed., Mack Publishing Company, Easton, Pennsylvania, 1995.

Thus, an immunogen described herein can be formulated with pharmaceutically acceptable carriers to help retain biological activity while also promoting increased stability during storage within an acceptable temperature range. Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

Formulated compositions, especially liquid formulations, may contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ^1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

The immunogenic compositions of the disclosure can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. The pharmaceutical composition may optionally include an adjuvant to enhance an immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AIPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the vaccine and cytokines, non-ionic block copolymers, and chemokines. Non- ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL- 12 (Genetics Institute, Cambridge, MA) may be used as an adjuvant (Newman et al. , 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

In some embodiments, the composition can be provided as a sterile composition. The pharmaceutical composition typically contains an effective amount of a disclosed immunogen and can be prepared by conventional techniques. Typically, the amount of immunogen in each dose of the immunogenic composition is selected as an amount which elicits an immune response without significant, adverse side effects. In some embodiments, the composition can be provided in unit dosage form for use to elicit an immune response in a subject, for example, to prevent HIV-1 infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof. In other embodiments, the composition further includes an adjuvant.

VII. Methods of Eliciting an Immune Response

The disclosed immunogens (e.g., a modified HIV-1 Env polypeptide, or a recombinant adenovirus expressing a modified Env polypeptide), polynucleotides and vectors encoding the disclosed immunogens, and compositions including same, can be used in methods of inducing an immune response to HIV-1 to prevent, inhibit, and/or treat an HIV-1 infection.

Provided herein are methods of eliciting an immune response against HIV-1 in a subject. In some embodiments, the method includes administering to the subject an effective amount of a modified Env polypeptide, Env trimer, recombinant adenovirus, nucleic acid molecule, vector or immunogenic composition disclosed herein. In some examples, the modified Env polypeptide, Env trimer, recombinant adenovirus, nucleic acid molecule, vector or immunogenic composition is administered intranasally (such as in a spray) or orally (such as by using enteric-coated tablets).

When inhibiting, treating, or preventing HIV-1 infection, the methods can be used either to avoid infection in an HIV-1 seronegative subject (e.g., by inducing an immune response that protects against HIV-1 infection), or to treat existing infection in an HIV-1 seropositive subject. The HIV-1 seropositive subject may or may not carry a diagnosis of AIDS. Hence, in some embodiments, the methods involve selecting a subject at risk for contracting HIV-1 infection, or a subject at risk of developing AIDS (such as a subject with HIV-1 infection), and administering a disclosed immunogen to the subject to elicit an immune response to HIV-1 in the subject.

To identify subjects for prophylaxis or treatment according to the methods of the disclosure, accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition, or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine environmental, familial, occupational, and other such risk factors that may be associated with the targeted or suspected disease or condition, as well as diagnostic methods, such as various ELISA and other immunoassay methods to detect and/or characterize HIV-1 infection. These and other routine methods allow the clinician to select patients in need of therapy using the methods and pharmaceutical compositions of the disclosure. In accordance with these methods and principles, a composition can be administered according to the teachings herein, or other conventional methods, as an independent prophylaxis or treatment program, or as a follow-up, adjunct or coordinate treatment regimen to other treatments.

The disclosed immunogens can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-HIV-1 immune response, such as an immune response to HIV-1 Env protein. Separate immunogenic compositions that elicit the anti-HIV-1 immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate immunization protocol.

In one embodiment, a suitable immunization regimen includes at least two separate inoculations with one or more immunogenic compositions including a disclosed immunogen, with a second inoculation being administered more than about two, about three to eight, or about four weeks following the first inoculation. A third inoculation can be administered several months after the second inoculation, and in specific embodiments, more than about five months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject's “immune memory.” The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively, the T cell populations can be monitored by conventional methods. In addition, the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of HIV-1 infection or progression to AIDS, improvement in disease state (e.g., reduction in viral load), or reduction in transmission frequency to an uninfected partner. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, a dose of a disclosed immunogen can be increased or the route of administration can be changed.

It is contemplated that there can be several boosts, and that each boost can be a different immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime.

The prime and the boost can be administered as a single dose or multiple doses, for example, two doses, three doses, four doses, five doses, six doses or more can be administered to a subject over days, weeks or months. Multiple boosts can also be given, such one to five, or more. Different dosages can be used in a series of sequential inoculations. For example, a relatively large dose in a primary inoculation and then a boost with relatively smaller doses. The immune response against the selected antigenic surface can be elicited by one or more inoculations of a subject.

In several embodiments, a disclosed immunogen can be administered to the subject simultaneously with the administration of an adjuvant. In other embodiments, the immunogen can be administered to the subject after the administration of an adjuvant and within a sufficient amount of time to elicit the immune response.

Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject, or that elicit a desired response in the subject (such as a neutralizing immune response). Suitable models in this regard include, for example, murine, rat, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer an effective amount of the composition (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the composition may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. The actual dosage of disclosed immunogen will vary according to factors such as the disease indication and particular status of the subject (for example, the subject’s age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of the composition for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.

A non- limiting range for an effective amount of the disclosed immunogen within the methods and immunogenic compositions of the disclosure is about 0.0001 mg/kg body weight to about 10 mg/kg body weight, such as about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, or about 10 mg/kg, for example, 0.01 mg/kg to about 1 mg/kg body weight, about 0.05 mg/kg to about 5 mg/kg body weight, about 0.2 mg/kg to about 2 mg/kg body weight, or about 1.0 mg/kg to about 10 mg/kg body weight. In some embodiments, the dosage includes a set amount of a disclosed immunogen such as from about 1-300 pg, for example, a dosage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 pg.

The dosage and number of doses will depend on the setting, for example, in an adult or anyone primed by prior HIV-1 infection or immunization, a single dose may be a sufficient booster. In naive subjects, in some examples, at least two doses would be given, for example, at least three doses. In some embodiments, an annual boost is given, for example, along with an annual influenza vaccination.

HIV-1 infection does not need to be completely inhibited for the methods to be effective. For example, elicitation of an immune response to HIV-1 with one or more of the disclosed immunogens can reduce or inhibit HIV-1 infection by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (elimination or prevention of detectable HIV-1 infected cells), as compared to HIV-1 infection in the absence of the therapeutic agent. In additional examples, HIV- 1 replication can be reduced or inhibited by the disclosed methods. HIV-1 replication does not need to be completely eliminated for the method to be effective. For example, the immune response elicited using one or more of the disclosed immunogens can reduce HIV-1 replication by a desired amount, for example, by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even 100% (elimination or prevention of detectable HIV-1 replication), as compared to HIV-1 replication in the absence of the immune response.

To successfully reproduce itself, HIV-1 must convert its RNA genome to DNA, which is then imported into the host cell's nucleus and inserted into the host genome through the action of HIV-1 integrase. Because the primary cellular target of HIV-1, CD4+ T-Cells, can function as the memory cells of the immune system, integrated HIV-1 can remain dormant for the duration of these cells' lifetime. Memory T-cells may survive for many years and possibly for decades. This latent HIV-1 reservoir can be measured by co-culturing CD4+ T-cells from infected patients with CD4+ T-cells from uninfected donors and measuring HIV-1 protein or RNA (See, e.g., Archin et al., AIDS, 22:1131-1135, 2008). In some embodiments, the provided methods of treating or inhibiting HIV-1 infection include reduction or elimination of the latent reservoir of HIV-1 infected cells in a subject. For example, a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% (elimination of detectable HIV-1) of the latent reservoir of HIV-1 infected cells in a subject, as compared to the latent reservoir of HIV-1 infected cells in a subject in the absence of the treatment with one or more of the provided immunogens.

Following immunization of a subject, serum can be collected from the subject at appropriate time points, frozen, and stored for neutralization testing. Methods to assay for neutralization activity, include, but are not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry based assays, single-cycle infection assays (e.g., as described in Martin et al. (2003) Nature Biotechnology 21:71-76), and pseudovirus neutralization assays (e.g., as described in Georgiev et al. (Science, 340, 751-756, 2013), Seaman et al. (J. Virol., 84, 1439-1452, 2005), and Mascola et al. (J. Virol., 79, 10103-10107, 2005), each of which is incorporated by reference herein in its entirety. In some embodiments, the serum neutralization activity can be assayed using a panel of HIV-1 pseudoviruses as described in Georgiev et al., Science, 340, 751-756, 2013 or Seaman et al. J. Virol., 84, 1439-1452, 2005. Briefly, pseudovirus stocks are prepared by co-transfection of 293T cells with an HIV-1 Env-deficient backbone and an expression plasmid encoding the Env gene of interest. The serum to be assayed is diluted in Dulbecco's modified Eagle medium- 10% FCS (Gibco) and mixed with pseudovirus. After 30 minutes, 10,000 TZM-bl cells are added, and the plates are incubated for 48 hours. Assays are developed with a luciferase assay system (Promega, Madison, WI), and the relative light units (RLU) are read on a luminometer (Perkin-Elmer, Waltham, MA). To account for background, a cutoff of ID50 > 40 can be used as a criterion for the presence of serum neutralization activity against a given pseudovirus.

In some embodiments, administration of an effective amount of one or more of the disclosed immunogens to a subject elicits a neutralizing immune response in the subject, wherein serum from the subject neutralizes, with an ID50 > 40, at least 10% (such as at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 70%) of pseudoviruses is a panel of pseudoviruses including the HIV-1 Env proteins listed in Table S5 or Table S6 of Georgiev et al. (Science, 340, 751-756, 2013), or Table 1 of Seaman et al. J. Virol., 84, 1439-1452, 2005).

One approach to administration of nucleic acids is direct immunization with plasmid DNA, such as with a mammalian expression plasmid. Immunization by nucleic acid constructs is taught, for example, in U.S. Patent No. 5,643,578 (which describes methods of immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or a humoral response), and U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637 (which describe operably linking a nucleic acid sequence encoding an antigen to regulatory sequences enabling expression). U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding immunogenic peptides or other antigens to an organism. The methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune-stimulating constructs, or ISCOMS™, negatively charged cage-like structures of 30-40 nm in size formed spontaneously on mixing cholesterol and Quil A™ (saponin). Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMS™ as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991). Doses of antigen as low as 1 pig encapsulated in ISCOMS™ have been found to produce Class I mediated CTL responses (Takahashi et al., Nature 344:873, 1990).

In some embodiments, a plasmid DNA vaccine is used to express a disclosed immunogen in a subject. For example, a nucleic acid molecule encoding a disclosed immunogen can be administered to a subject to elicit an immune response to HIV-1 gpl20. In some embodiments, the nucleic acid molecule can be included on a plasmid vector for DNA immunization, such as the pVRC8400 vector (described in Barouch et al., J. Virol, 79, 8828-8834, 2005, which is incorporated by reference herein). In another approach to using nucleic acids for immunization, a disclosed immunogen (such as a modified HIV-1 Env polypeptide) can be expressed by attenuated viral hosts or vectors or bacterial vectors. Recombinant vaccinia virus, adeno- associated virus (AAV), herpes virus, retrovirus, cytomegalovirus or other viral vectors can be used to express the peptide or protein, thereby eliciting a CTL response. For example, vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848. BCG (Bacillus Calmette Guerin) provides another vector for expression of the peptides (see Stover, Nature 351:456-460, 1991).

In one embodiment, a nucleic acid encoding a disclosed immunogen (such as a modified HIV-1 Env polypeptide, or a recombinant adenovirus expressing a modified Env polypeptide) is introduced directly into cells. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 Jlg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

Example 1: Materials and methods

This example describes the materials and experimental procedures for the studies described in Example 2.

Construction of Env designs in a plasmid shuttle vector

HIV-1 strain 1086 clade C Env non-stabilized and stabilized constructs were designed. All 134 Env designs included the K160N point mutation (KN) and were synthesized in a plasmid shuttle vector driven by a CMV promoter, unless otherwise noted. Most plasmid shuttle vectors were designed for insertion into the adenoviral E3 region and included the endogenous E3 polyadenylation signal (poly A) (Alexander et al., PLoS One 7(2):e31177, 2012). Some plasmid shuttle vectors were designed for insertion at the end of the adenoviral E3 region and included an exogenous bovine growth hormone (BGH) poly A immediately after the Env gene (End-E3) (FIG. 9).

Env truncations: The 1086C Env was designed with or without truncations of the gp41 cytoplasmic tail corresponding to gpl60, gpl55, gpl50, gpl48, and gpl45. For each truncation length, a non- stabilized Env with a K160N mutation was designed as well as a stabilized Env with a K160N mutation, the SOSIP stabilization, and a 6R enhanced furin cleavage site (6R) (Table 2). The non- stabilized gpl50 K160N Env was also synthesized in an End-E3 plasmid shuttle vector.

Table 2. Env mutation abbreviations

Cleavage incompetent Env: EnvC150mut, a furin cleavage incompetent Env without the K160N point mutation from a prior clinical trial, was compared with other Env designs (Alexander et al., PLoS ONE 8(12):e82380, 2013). A K160N point mutation was introduced to produce EnvC150mut-KN. Both the EnvC150mut and EnvC150mut-KN plasmids were driven by the native adenoviral major late promoter (MLP) with a BGH poly A immediately after the Env gene.

Native flexible linker Env: Three Env constructs were designed with a native flexible linker (NFL) of G4SG4S (SEQ ID NO: 13) that obviated the need for furin- mediated cleavage (Sharma et al., Cell Rep 11 (4):539-50, 2015). Each construct had an additional G4SK (SEQ ID NO: 10) flexible peptide linker immediately before the transmembrane region. Each construct had a series of previously described stabilizing BG505-trimer derived (TD) mutations and an I559P point mutation (IP) (Guenaga et al., J Virol 90(6):2806-17, 2015) as well as these stabilizing mutations N302Y, L519R, F520R, L568G and N636G (Guenaga et al., Immunity 46(5):792-803, 2017). One Env construct was truncated to gpl46 and the native Env signal peptide was replaced with the signal peptide of CD5 to produce Envl46-NFL-TD-CD5. The plasmid for this construct contained a HTLV-1 R region containing a splice donor and a CMV IE splice acceptor. Another Env construct was truncated to gpl45 to produce Envl45-NFL-TD. The Envl45-NFL-TD also had an AENL motif inserted after the signal peptide. Finally, this construct was also designed with the signal peptide of CD5 to produce Envl45-NFL-TD-CD5.

SOSIP.v5.2.8: An Env construct named Envl45-SOSIP.v5.2.8 was designed. The Env construct was truncated to gpl45, and the wild type Env signal peptide was replaced with the tissue plasminogen activator (tPA) signal peptide. The wild type furin cleavage site was replaced with 6R to increase furin cleavage. Stabilizing mutations and disulfide bonds were introduced from the previously described SOSIP.v5.2 (Torrents de la Pena et al., Cell Reports 20(8):1805-1817, 2017).

Interdomain Stabilized Env: Three Env constructs were designed with a stabilizing disulfide bond between the gpl20 inner and outer domains (Zhang et al. , Cell Host Microbe 23(6):832-44.e6, 2018). All constructs were truncated to gpl50. Each construct contained either a I109C or a D113C mutation and either an E429GCG or a R432GCG mutation. The 3 constructs were named Envl50-I109C-E429GCG, Envl50-D113C-E429GCG, and Envl50-D113C- R432GCG.

Additional stabilized Env: Six Env constructs were designed with various combinations of stabilizing mutations. Constructs were either truncated to a gpl50 or the N and C terminus of gpl20 as well as gp41 were from the BG505 (clade A) sequence (chimera). Constructs contained either a 6R enhanced cleavage site or a single chain G3SG4SG2 (SEQ ID NO: 14) linker that replaced the wild type furin cleavage site (sclOln). All constructs had an A433P point mutation. Additional mutations included the SOSIP stabilization; the N302M, T320L, and A330P stabilizing point mutations (3mut); and the L154M, N300M, N302M, and T320L stabilizing point mutations (4mut). In some designs, the Env transmembrane region was replaced with an influenza hemagglutinin transmembrane region (HATM). In some designs, the MPER epitope was replaced with a G2SG2SG3S linker (SEQ ID NO: 7) (noMPER). Some designs included a His tag (His). The 6 Env constructs were named Envl50-6R-A433P (End-E3), Envl50-6R-A433P-3mut-His, Envl50- 6R-A433P-4mut-His, Env-chimera-SOSIP-scl01n-noMPER-HATM-A433P (End-E3), Env- chimera-SOSIP-scl01n-noMPER-HATM-A433P-3mut-His, and Env-chimera-SOSIP-sclOln- noMPER-HATM- A433P-4mut-His .

Stabilized Env for down-selection: An additional 108 stabilized Env constructs were designed for down- selection before further analysis. Some of the stabilized constructs contained a 6R enhanced cleavage site, contained a disulfide bond within gpl20 from the I201C and A443C point mutations (DS), and had an HATM. Each Env construct had one of the following designs: (1) the native 1086C sequence, (2) the chimera sequence (3) the chimera sequence plus rare 1086C strain-specific residues were substituted with more prevalent ones (repair), or (4) the chimera and repair sequence plus stabilizing mutations that optimize regions of Env folding (stabilize) (Rutten et al., Cell Reports 23(2):584-595, 2018). Additionally, each Env construct had either no point mutations, the 3mut mutations, or the 4mut mutations. Each Env construct included or did not include the T569G and N636G point mutations (2G) (Guenaga et al., Immunity 46(5):792-803, 2017). Each Env construct included either the SOSIP stabilization or only the IP mutation.

Finally, each Env construct included either an unedited MPER epitope (withMPER) or the MPER epitope was replaced with a linker (noMPER). Every combination of each of these categories was constructed to produce 96 Env designs. Eight additional Env designs were constructed with the above criteria but no DS, 4mut, 3mut, 2G, or SOSIP conditions. Two of the interdomain stabilized Env constructs listed above were also truncated to a gpl45, and one of these constructs also had R503GCG and T605C point mutations added. The Envl45-NFL-TD construct had the interdomain stabilizing I109C and E429GCG mutations introduced.

Down-selection of stabilized Env constructs

A cell surface membrane protein ELISA was performed to down-select the 108 stabilized Env constructs. Binding of antibodies CAP256-VRC26.25, PGT145, 17b, 17b with CD4, 447- 52D, and F105 was tested by ELISA. Env designs were ranked by the CAP256-VRC26.25 times PGT145 binding values, and Env designs with 17b, 17b with CD4, F105, and 447-52D binding values above 0.2 were excluded. Two Env constructs were down-selected using this metric: Envl45-repair-DS-IP-6R-noMPER-HATM-3mut-2G and Envl45-repair-DS-SOSIP-6R- withMPER-HATM-4mut.

The 108 stabilized Env constructs were also down-selected by a flow cytometric analysis of broadly neutralizing antibody (bnAb) binding. One day prior to transfection, 1 million A549 adenocarcinoma cells (ATCC, CCL-185) were seeded in T-25 flasks with F12K media (ATCC, cat. 30-2004) containing 1% penicillin-streptomycin-glutamine (PSG, Gibco, cat. 10378016), and 10% fetal bovine serum (FBS, Gemini Bio-Products, cat. 100-106) at 37°C and 5% CO2. Cells were transfected by adding 5 pg of plasmid DNA, 25 pl of DNA-IN (MTI-GlobalStem, cat. 73772), and 50 pl of Opti-MEM (Gibco, cat. 31985070).

Cells were collected 2 days post-transfection with 0.01 M EDTA in phosphate buffered saline (PBS, Quality Biological, cat.114-058-101). Env expression and antigenicity were measured by cell-surface staining of Env with 50 pl of antibodies VRC01, PGT151, F105, and 447-52D diluted to a concentration of 1 pg/ml with PBS containing 1% HEPES (Gibco, cat. 15630080) and 0.09% bovine serum albumin (BSA, Sigma-Aldrich, cat. A7979) for 1 hour at 37°C. Cells were then stained with 100 pl of a secondary antibody, goat anti-human IgG Fab2-PE (Jackson Immuno Research, cat. 109-116-097) at a 1:100 dilution for 1 hour at 37°C. Cells were also stained with 100 pl of live/dead fixable violet dead cell stain (Invitrogen, cat. L34964) at a 1:250 dilution for 30 minutes at room temperature. Cells were fixed with 250 pl of Cytofix/Cytoperm (BD, cat. 554722) for 20 minutes on ice. Cells were stored at 4°C overnight and 10⁵ cells per sample were analyzed by flow cytometry on a BD FACS Aria.

Five Env constructs were down-selected by this metric: Envl45-repair-IP-6R-noMPER- HATM, Env-chimera-IP-6R-noMPER-HATM, Env-chimera-DS-SOSIP-6R-withMPER-HATM- 3mut-2G, Env-chimera-DS-SOSIP-6R-noMPER-HATM-4mut, and Env-chimera-DS-SOSIP-6R- noMPER-HATM-3mut-2G.

Flow cytometric analysis of Env antigenicity expressed by the shuttle vector

The 7 down-selected Env constructs and all other Env constructs were analyzed by an expanded antibody panel. One day prior to transfection, 4.5 million A549 cells were seeded in T-75 flasks as above. Cells were transfected with 15 pg of plasmid DNA, 75 pl of DNA-IN, and 150 pl of Opti- MEM. Cells were stained as before with an expanded antibody panel of PGT145, PG16, VRC01, bl2, PGT151, 8ANC195, 35022, 10E8, F105, and 447-52D at 1 pg/ml. By this metric, 19 Env constructs were selected for recombination in an Ad4 vector.

Construction, production, and purification of Ad4-Env recombinant viruses

Bacterial homologous recombination was used to generate a plasmid with both the complete Ad4 genome from the Ad4 military vaccine (Accession AY594254) and various down-selected Env designs as previously described (Alexander et al., PLoS One 7 (2):e31177 , 2012). The shuttle vector containing the various Env designs was inserted within the Ad4 E3 region, which included either a partial deletion of E3 (PDE3) or a full deletion of E3 (FDE3). The PDE3 removed the E3 24.8K, 6.8K, and 29.7K ORFs while the FDE3 removed the E3 23.3K, 19K, 24.8K, 6.3K, 29.7K, 10.4K, 14.5K, and 14.7K ORFs (FIG. 9). For additional Ad4-Env recombinants, Env genes were inserted after the E3 region with the same partial deletion of E3 (PDE3-End). Both the FDE3 and PDE3-End designs included a BGH poly A immediately after the Env gene.

Homologous recombination was performed in BJ5183 recombination-competent bacteria cells (Agilent, cat. 200154) as previously described (Alexander et al., PLoS One 7 (2):e31177 , 2012). Clones were screened by PCR using the forward primer 5’- AGCTCTTCACTGGGTTTGCGAC-3’ (SEQ ID NO: 15) and the reverse primer 5’- TTCAGATCCCGTGGATCTGG-3’ (SEQ ID NO: 16), which span the upstream homologous recombination site. PCR-positive clones were screened by restriction enzyme digestion and transformed into DH10B bacterial cells (Invitrogen, cat. 18290015) for DNA production. Clones were again screened by restriction enzyme digestion and recombination was confirmed by DNA sequencing.

Recombinant Ad4-Env virus was produced by linearizing recombinant DNA and transfecting A549 cells. One day prior to transfection, 2 million A549 cells were seeded in a 10 cm dish (Stellar Scientific, cat. TC-D0100). Ten pg of recombinant DNA was linearized with PacI and Opti-MEM was added up to a total volume of 1 ml. Linearized DNA was mixed with 40 pl of FuGENE HD Transfection Reagent (Promega, cat. E2312) and incubated at room temperature for 15 minutes before transfection of A549 cells. Three days post-transfection, cells were collected with 0.01 M EDTA in PBS and transferred to three T-225 flasks. Cells were incubated for 7 to 14 days until cytopathic effect (CPE) was observed and an Adeno Test kit (SA Scientific, cat. 067020) demonstrated adenovirus production. The primary viral stock was harvested by scraping cells to detach. Cells were lysed by three cycles of freezing in a dry ice and ethanol bath and then thawing. The lysed cell solution was clarified by centrifugation at 1000 rpm for 10 minutes at 4°C. The lysed cell supernatant and the original cell culture supernatant were combined, flash frozen in a dry ice and ethanol bath, and stored at -80°C.

A 10-layer cell chamber was seeded with 32 million A549 cells and incubated for 4 days. Cells were washed with 500 ml of Dulbecco’s phosphate buffered saline (DPBS, Sigma- Aldrich, cat. D8537), infected with between 5 x 10⁹ and 2.5 x 10¹² Ad4-Env recombinant virus particles, and cultured in serum free media containing 1% PSG. Viral growth was monitored with the Adeno Test kit. Virus was harvested 3 to 7 days post-infection with a Triton X-100, MES monohydrate detergent buffer. Expanded virus was purified by size-exclusion filtration, anion exchange chromatography, and ultrafiltration using the KR2i Tangential Flow Filtration System (Spectrum).

An enzyme-linked immunoassay with the AdEasy Viral Titer Kit (Agilent Technologies, cat. 972500) was used to titer the concentration of infectious virus particles in the purified virus solution. Human embryonic kidney (HEK) 293T cells (ATCC, CRL-3216) (2.2 x 10⁵) were incubated with 50 pl of purified virus serially diluted from 10’² to 10’⁶ in high glucose Dulbecco’s Modified Eagle Media (DMEM, Gibco cat. 11965092) containing 1% PSG and 10% FBS at 37°C and 5% CO2 for 48 hours. Cells were fixed with methanol and then sequentially stained with an anti-hexon antibody, a horseradish peroxidase-conjugated antibody, and 3,3’diaminobenzidine (DAB) substrate, which produced a dark brown precipitate. The number of dark brown cells was used to calculate the concentration of infectious units (IFU). The viral particle titer was determined by HPLC.

Flow cytometric analysis of Env antigenicity expressed by Ad4 vector

To measure Env expression and antigenicity in the Ad4 vector, A549 cells were infected with the Ad4-Env recombinants and the binding of a panel of bnAbs was analyzed. One day prior to infection, 4.5 million A549 cells were seeded in T-75 flasks as above. Cells were infected with each of the Ad4-Env recombinants at a MOI of 0.1 to 1. Env cell surface staining was performed as before with the 10 antibody panel. EnvC150mut, Envl50, and Envl50-NFL-TD were also stained with VRC34. Cells were permeabilized overnight in Perm/Wash buffer (BD, cat. 554723). Cells were intracellularly stained with 50 pl of an anti-hexon (adenoviral capsid protein) antibody 8C4-APC (Novus, cat. NB600-413APC) at a 1:700 dilution in Perm/Wash buffer for 30 minutes on ice (FIGS. 10A-10D).

Electron microscopy

A 10-chamber cell stacker was seeded with A549 cells as before and infected with Ad4-Env recombinant viruses at a MOI of approximately 0.5. Cells were harvested using 0.01 M EDTA in PBS 2 days post-infection. Cell surface Env was extracted from the membrane with 3BNC117.

Negative-stain transmission electron microscopy was performed to confirm trimer conformation. The samples were diluted to 10-20 pg/ml with a buffer containing 10 mM HEPES, pH 7, and 150 NaCl. A 4.7 pl drop was applied to a glow-discharged carbon-coated copper grid. After 15 seconds, the drop was removed, and the grid was washed with the same buffer, followed by negative staining with 0.7% uranyl acetate. Micrographs were collected using SerialEM (Mastronarde, J Struct Biol 180(3):519-530, 2012) on an FEI Tecnai T20 microscope operated at 200 kV and equipped with a 2k x 2k Eagle CCD camera (pixel size: 2.2 A/pixel) or using the EPU software on a Thermo Scientific Tales F200C G2 microscope operated at 200 kV and equipped with a 4k x 4k Ceta CCD camera (pixel size: 2.5 A/pixel). Particles were selected from the micrographs automatically using in-house written software and subjected to reference-free 2D classification in Relion (Scheres, J Struct Biol 180(3):519-530, 2012).

Rabbit immunization

Immunogenicity of Ad4-Env recombinants was tested by immunizing female New Zealand White rabbits. Groups of 6 rabbits were immunized with 10¹¹ Ad4-Env recombinant virus particles on weeks 0 and 4. Rabbits were intramuscularly immunized in the quadricep, with 0.5 ml delivered to each hind leg. As a negative control, 6 rabbits were immunized with 10¹¹ wild type Ad4 virus particles following the same schedule. Blood was collected prior to immunization on weeks 4, 8, and 12. Rabbits immunized with Ad4-FDE3-Envl50 and Ad4-PDE3-Envl45-NFL-TD-CD5 were also boosted with 30 pg of a heterologous soluble trimeric protein VRC-HIVRGP096-00-VP (Trimer 4571) with alum based upon the clade A BG505 Env strain. Blood from these rabbits was also collected on weeks 14, 16, and 20.

Neutralizing activity of rabbit serum was measured by a pseudovirus entry inhibition assay using HIV-1 strains 1086C and SF162. HIV-1 Env pseudoviruses were generated by cotransfection of 293T cells with an Env-deficient backbone (pSG3AEnv) and a second plasmid that expressed HIV-1 Env at a ratio of 3:1. Seventy-two hours after transfection, supernatants containing pseudoviruses were harvested and frozen at -80°C until further use. As described previously, heat- inactivated rabbit serum was serially diluted five- fold with Dulbecco’s modified Eagle medium containing 10% FCS (Gibco), and 10 pl of serum or mAb was incubated with 40 pl of pseudovirus in a 96-well plate at 37°C for 30 minutes (Li et al., J Virol 79(16):10108-10125, 2005). TZM-bl cells were then added and plates were incubated for 48 hours. Assays were then developed with a luciferase assay system (Promega, Finnboda Varvsvag, Sweden), and the relative light units (RLU) were read on a luminometer (Perkin Elmer).

Example 2: Evaluation of stabilized and non-stabilized HIV-1 envelope expressed by a replication competent Ad4 vector

This example describes studies undertaken to develop Env designs that provide a native-like conformation.

Truncation of gp41

Initial studies examined the impact on variations in the length of the HIV Env cytoplasmic tail on the surface expression level and confirmation of Env. Prior studies demonstrated that the cytoplasmic tail can be toxic to cells. In addition, the cytoplasmic tail contains a clathrin binding endocytosis motif and other sequences that mediate a reduction in cell surface expression. Ten HIV-1 1086c non-stabilized and S OSIP-stabilized Env length variants were designed by truncating gpl60 to form gpl55, gpl50, gpl48, or gpl45 (FIG. 1A). There was a progressive increase in cell surface expression of the non-stabilized and SOSIP-stabilized Env as measured by the binding of VRC01 that was greatest in the gpl45 truncations (FIG. IB). This is consistent with the removal of either known or other less well characterized sequences that mediate endocytic functions. The antigenicity of cell-surface expressed Env was measured by staining with a panel of 10 antibodies that bound 5 epitopes: the V1-V2 loops at the trimer apex (PGT145, PG16), the V3 loop (447- 52D), the CD4 binding site (VRC01, bl2, F105), the gpl20-gp41 interface (PGT151, 8ANC195, 35022), and the membrane-proximal external region (10E8). Truncation of gp41 had minimal effects on the Env antigenicity (FIG. 1C).

Antigenicity

Additional Env constructs were designed to stabilize Env in a closed formation (FIG. 2). Env constructs were down-selected by two metrics. First, two Env constructs were selected by a cell surface membrane protein ELISA. Env constructs were ranked by the CAP256-VRC26.25 times PGT145 binding values, and Env designs with 17b, 17b with CD4, F105, and 447-52D binding values above 0.2 were excluded (FIG. 11). Second, five Env constructs were selected by a flow cytometric analysis. Cell surface expressed Env constructs were stained with VRC01, PGT151, F105, and 447-52D. Constructs were ranked by PGT151 binding, and five constructs with low F105 and 447-52D binding as well as high cell surface expression as measured by VRC01 were selected (FIG. 12).

An expanded panel of 10 antibodies was used to measure the antigenicity of 24 nonstabilized and stabilized Env constructs expressed on the cell surface by transfection of a plasmid vector. The cleavage incompetent EnvC150mut failed to bind bnAbs PGT145, PG 16, and PGT151 (FIG. 3). Introduction of the K160N point mutation in EnvC150mut-KN restored binding of apexbinding bnAb PG 16. Most Env constructs showed good cell surface binding of PGT145, PG 16, bl2, and PGT151 in contrast to EnvC150mut. All constructs did not bind or weakly bound 8ANC195, 35022, and 10E8 as expected. Most Env constructs bound F105 and 447-52D, indicating at least a portion of the Env molecules were in a non-native-like conformation. However, Env constructs with the native flexible linker as well as Envl45-SOSIP.v5.2.8 eliminated binding of antibodies F105 and 447-52D, demonstrating the most native-like conformation. The native flexible linker blocked the gpl20-gp41 interface epitope; therefore, these Env constructs did not bind PGT151.

Nineteen Env constructs were recombined with an Ad4 backbone to produce live, replication-competent Ad4-Env recombinants. The antigenicity of Env expressed by infection of cells with the viral recombinants was characterized as before with the panel of 10 antibodies. Cells were also stained with an anti-hexon (adenoviral capsid protein) antibody to confirm that Env expression was specific to virus-infected cells. A similar antibody binding profile was observed as with the plasmid vector (FIG. 4B). Ad4-Envl50 and Ad4-Envl45-NFL-TD-CD5 were additionally stained with VRC34, which binds the fusion peptide epitope. The non- stabilized Envl50 bound VRC34; however, the native flexible linker blocked binding to the fusion peptide epitope in Ad4- Envl45-NFL-TD-CD5 (FIG. 4A).

Env conformation was further confirmed by negative stain electron microscopy (EM) of eight Env designs. Env was extracted from the cell membrane with 3BNC117; therefore, the Env transmembrane regions tended to produce large aggregates, trimers of Env trimers, and dimers of Env trimers (FIG. 5). The EnvC150mut did not form native trimers; however, 2D classifications showed loose trimeric objects. Envl50 showed no Env trimers by EM, yet this construct was nonstabilized and had no permanent linker between the gpl20 and gp41 subunits. Other stabilized Env constructs including Envl45-NFL-TD-CD5 and Envl45-SOSIP.v5.2.8 showed Env trimers with bound antibody in a native-like conformation.

Immunogenicity

Eighteen Ad4-Env recombinants with the most favorable characteristics of expression and confirmation were selected to test immunogenicity in rabbits. Rabbits were used for this purpose because of prior experience in testing Ad4 recombinants in this model and published data on the immunogenicity of stabilized Env trimers in this model. In addition to the autologous tier 2 strain 1086C, neutralizing was also measured against the heterologous tier 1 strain SF162, which adopts an open conformation and is readily neutralized by V3-specific antibodies. The Ad4-EnvC150mut induced no neutralization against 1086C and low neutralization against SF162 (FIG. 6), consistent with its non-native-like conformation. Serum taken at week 12 from rabbits immunized with the non-stabilized Ad4-Envl50 induced autologous neutralization of 1086C pseudovirus (median ID50 = 182) and neutralization of SF162 pseudovirus (median ID50 = 26). Immunization with the stabilized Ad4-Envl45NFL-TD induced similar levels of neutralization of the autologous 1086C (median ID50 = 264). However, consistent with its lack of exposure of CD4i epitopes, Ad4- Envl45-NFL-TD induced no neutralization against SF162 at week 12. A similar pattern was observed for two other stabilized designs, Ad4PDE3 Envl45-NFL-TD-CD5 and Ad4PDE3 Envl45-SOSIP.v5.2.8.

Two soluble Env trimers were being developed for clinical use that could potentially be used to boost antibody responses in a clinical trial. The first of these is the BG505 (clade A) DS- SOSIP soluble trimer (VRC-HIVRGP096-0, Trimer 4571) that in addition to the SOSIP mutations also has an additional stabilizing mutation by a covalent linkage at between residues 201 and 433 (Liu et al., Nat Struct Mol Biol 24(4):370-378, 2017). The second is the 16055 (clade C) degly-4, which lacks the 4 N-linked glycosylation sites that surround the CD4 binding site. It is thought that the absence of a glycan at position 276 in the Env of the virus may have played a role in the development of the well-known CD4 binding site antibody VRC01 in the patient from whom it was isolated. It was therefore thought that elimination of some or all of these glycosylation sites may help to initiate CD4 binding site targeted neutralizing antibodies. All five animal groups were boosted with the BG505 DS-SOSIP trimer at 12 weeks and the 16055 DeGly4 at 20 weeks. There was no significant difference in the levels of neutralizing antibodies against the 1086 strain in animals that received Ad4-FDE3-Envl50 with or without boosting with trimers (FIG. 7).

Next, the breadth of responses at week 28 was examined. In addition to neutralizing activity to SF162 and the homologous 1086C, animals that received Ad4-FDE3-Envl50 without a trimer boost did develop some sporadic and low level neutralization of the Tier 3 virus PVO.04 and the Tier 2 virus Q769.d22 (FIG. 13). A similar pattern was observed in animals that received Ad4- FDE3-Envl50 with subsequent trimer boosts, however the neutralizing activity was higher in magnitude and also included Dul56.12 and ZM106.9. Neutralization induced by the Ad4PDE3 Envl45-NFE-TD-CD5 was limited to the autologous 1086C strain and only low level neutralization was observed against the BG505 strain used in the DS-SOSIP trimer. Taken together these results indicate that Ad4 expressing 1086C can induce neutralizing antibodies and that, in the case of the non- stabilized design, can be broadened by boosting, particularly in response to the 16055 DeGly4 trimer.

Discussion

The results of the present study indicate that several changes in the design of Env immunogens presented by Ad4 can result in the induction of neutralizing antibodies and that these responses can be broadened by boosting with stabilized trimers. Among the designs that were tested, the change with the largest impact on the induction of neutralizing antibodies was the restoration of the furin cleavage site. This change resulted in the restoration of binding by bnAbs and the induction of autologous 1086c neutralizing antibodies in rabbits. In one prior study, Ad4- Envl50 and Ad4-Envl45-NFE-TD-CD5 were shown to be immunogenic when administered intramuscularly in nonclinical rabbit studies, inducing serum neutralizing antibodies capable of neutralizing 1086 pseudovirus, and afforded some protection against SHIV exposure in rhesus macaques vaccinated with a prime-boost regimen of intranasal Ad4-Envl50KN followed by an IM 1086 protein antigen (Malherbe et al., J Virol 92(2): e01092-17, 2018).

In addition to testing whether either or both of these designs induce neutralizing antibodies, their relative immunogenicity to each other was tested. Both of these designs have advantages (FIG. 8). The Ad4-Envl50KN does not have stabilizing mutations and therefore has sufficient flexibility to assume multiple conformation states important to allow binding of gp 120-41 interface and membrane-proximal external region (MPER) antibodies. The Env expressed by Ad4- Envl45NFL, through its stabilizing mutations and lack of a need for proteolytic cleavage, presents the immune system with Env forms that have more uniformity. However, this Env cannot assume post-CD4-binding conformations and in theory will not permit the binding of interface or MPER- specific B cell antigen receptors in the context of cellular membranes. This Env also eliminates exposure of the V3 loop. In this vaccination context, V3 directed antibodies are thought to mediate neutralization of SF162 pseudo viruses, in which Env adopts a more open conformation. The elimination of V3-mediated neutralization in animals immunized with Ad4-Envl45NFL-TD supports that this design results in a closed conformation. Therefore, one non-stabilized and one stabilized design are being clinically tested. These inserts serve as model antigens to evaluate the replicating Ad4 platform and may be substituted in future trials should better candidates become available.

Participants will receive a booster vaccine consisting of a heterologous soluble trimeric protein VRC-HIVRGP096-00-VP (Trimer 4571) with alum, based upon the clade A BG505 Env strain. This is a protein developed to have stabilizing mutations and disulfide bonds, and engineered to be specifically recognized by broadly neutralizing antibodies and resist conformational change caused by CD4 binding. In previous studies with Ad4-H5-Vtn, boosting with protein antigens consistently provided the highest serum neutralizing antibody responses (Gurwith et al., Lancet Infect Dis 13(3):238-250, 2013; Khurana et al., PLoS One 10(l):e0115476, 2015; Patterson and Robert- Guroff, Expert Opin Biol Ther 8(9):1347-1363, 2008). The VRC- HIVRGP096-00-VP soluble trimer and others like it are considered the soluble protein antigens with the best prospects for inducing neutralizing antibodies among those currently available for clinical use.

There are a number of theoretical reasons that live, replication-competent adenovirus recombinant vaccines may be even more immunogenic than replication-incompetent vectors in humans. First, there is the potential for greater total antigen dose. Live vectors may replicate to levels that exceed the total dose of replication- incompetent vectors. In addition, they provide prolonged expression of inserted genes, which is likely critical to the development of effective cellular or humoral immunity. Importantly, this expression persists until it is terminated by an effective immune response. Live vectors also induce proinflammatory cytokines and costimulatory molecules that function as adjuvants to improve immunogenicity. Because these vectors replicate in the epithelium of the upper respiratory tract and gut, they offer the potential to induce cellular and humoral mucosal immune responses. Lastly, live, replication-competent adenovirus vectors may more readily overcome subtype-specific immunity to the vector itself. Upper respiratory tract inoculation of adenoviruses to seropositive humans does result in reinfection. The results with Ad4-H5-Vtn or HIV recombinants have been consistent with this experience. This property offers the potential for boosting in humans that may have received a prior dose of an adenovirus recombinant.

There are also a number of reasons that live Ad4 makes a particularly attractive recombinant vector. Wild-type Ad4 vaccine has been used for over 25 years in the military, and has been proven safe and effective at preventing acute respiratory disease in recruits. These vaccines have an extraordinary safety record and have been given to more than 10 million people (Gaydos and Gaydos, Mil Med 160(6):300-304, 1995). In addition, there are extensive clinical data on the safety, duration of shedding, transmissibility, and immunogenicity of these viruses. The Ad4 vaccine used by the military is a non-attenuated wild-type virus that is administered in an enteric- coated tablet. When administered via the gastrointestinal tract, the virus causes a selective enteric infection that is immunogenic and efficacious but does not spread to the upper respiratory tract or cause disease. These vaccines are easy to administer as enteric-coated tablets, inexpensive, and stable. While prevalence in humans of Ad5 is significant, one report indicated that following cessation of the Ad4 and Ad7 military vaccine program in 1996, 66% and 73% of new military recruits lacked antibodies considered protective against Ad4 and Ad7, respectively. Overall, 88% had no evidence of protective immunity to either Ad4 or Ad7 (Ludwig et al. , J Infect Dis 178(6): 1776-1778, 1998). Although pre-existing immunity to Ad4 or Ad7 may diminish the immunogenicity of these vectors, reinfection of seropositive individuals does occur. In addition, replication-competent Ad5-SIV recombinants have shown considerable promise in rhesus macaques (Patterson and Robert- Guroff, Expert Opin Biol Ther 8(9): 1347-1363, 2008). Thus, the Ad4 vector is an appropriate choice for development of HIV recombinants.

In view of the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.

Claims

1. A recombinant replication competent adenovirus type 4 (Ad4) expressing a modified HIV-1 clade C strain 1086 (1086C) envelope (Env) polypeptide, wherein the modified Env polypeptide comprises:

(i) a native HIV-1 signal peptide at the N-terminus; an asparagine substitution at position 160, numbered with reference to the HXB2 numbering scheme; a cleavage competent sequence positioned between the gpl20 and gp41 subunits; and a C-terminal truncation that results in deletion of a portion of the cytoplasmic domain; or

(ii) a heterologous CD5 signal peptide sequence at the N-terminus; a plurality of amino acid substitutions to stabilize Env trimer formation; an asparagine substitution at position 160; a proline substitution at position 559; a first heterologous peptide linker positioned between the gpl20 and gp41 subunits; a second heterologous peptide linker positioned between the ectodomain and the transmembrane domain; and a C-terminal truncation that results in deletion of the cytoplasmic domain, wherein the amino acid positions are numbered with reference to the HXB2 numbering scheme.

2. The recombinant replication competent Ad4 of claim l(i), wherein the cleavage competent sequence comprises residues 487-497 of SEQ ID NO: 6.

3. The recombinant replication competent Ad4 of claim l(i) or claim 2, wherein the C- terminal truncation results in an Envl50 polypeptide.

4. The recombinant replication competent Ad4 of claim l(i), claim 2 or claim 3, wherein the amino acid sequence of the Env polypeptide is at least 95% identical to SEQ ID NO: 6.

5. The recombinant replication competent Ad4 of any one of claim l(i) and claims 2-4, wherein the amino acid sequence of the Env polypeptide comprises or consists of SEQ ID NO: 6.

6. The recombinant replication competent Ad4 of claim l(ii), wherein the CD5 signal peptide sequence comprises residues 1-24 of SEQ ID NO: 3.

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7. The recombinant replication competent Ad4 of claim l(ii) or claim 6, wherein the plurality of amino acid substitutions comprises:

(a) an aspartic acid substitution at position 47 ;

(b) a glutamic acid substitution at position 49;

(c) a lysine substitution at position 65;

(d) a threonine substitution at position 106;

(e) an arginine substitution at position 166;

(f) a glutamine substitution at position 170;

(g) a tyrosine substitution at position 302;

(h) a methionine substitution at position 320;

(i) an arginine substitution at position 429;

(j) a glutamine substitution at position 432;

(k) an arginine substitution at position 500;

(l) a tyrosine substitution at position 513;

(m) an arginine substitution at position 519;

(n) an arginine substitution at position 520;

(o) a glycine substitution at position 568;

(p) a glycine substitution at position 636; or

(q) a combination of two or more of (a) to (p), wherein the amino acid position is numbered with reference to the HXB2 numbering scheme.

8. The recombinant replication competent Ad4 of claim 7, wherein the plurality of amino acid substitutions comprises the combination of (a) to (p).

9. The recombinant replication competent Ad4 of any one of claim 1 (ii) and claims 6-

8, wherein the first heterologous peptide linker comprises the amino acid sequence G4SG4S (SEQ ID NO: 13).

10. The recombinant replication competent Ad4 of any one of claim 1 (ii) and claims 6-

9, wherein the second heterologous peptide linker comprises the amino acid sequence G4SK (SEQ ID NO: 10).

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11. The recombinant replication competent Ad4 of any one of claim 1 (ii) and claims 6-

10, wherein the C-terminal truncation results in an Envl45 polypeptide.

12. The recombinant replication competent Ad4 of any one of claim 1 (ii) and claims 6-

11, wherein the amino acid sequence of the Env polypeptide is at least 95% identical to SEQ ID NO: 3.

13. The recombinant replication competent Ad4 of any one of claim 1 (ii) and claims 6-

12, wherein the amino acid sequence of the Env polypeptide comprises or consists of SEQ ID NO: 3.

14. The recombinant adenovirus of claim 1, wherein the genome of the adenovirus comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

15. A modified HIV-1 clade C strain 1086 (1086C) envelope (Env) polypeptide, wherein the modified Env polypeptide comprises:

(i) a native HIV-1 signal peptide at the N-terminus; an asparagine substitution at position 160, numbered with reference to the HXB2 numbering scheme; a cleavage competent sequence positioned between the gpl20 and gp41 subunits; and a C-terminal truncation that results in deletion of a portion of the cytoplasmic domain; or

(ii) a heterologous CD5 signal peptide sequence at the N-terminus; a plurality of amino acid substitutions to stabilize Env trimer formation; an asparagine substitution at position 160; a proline substitution at position 559; a first heterologous peptide linker positioned between the gpl20 and gp41 subunits; a second heterologous peptide linker positioned between the ectodomain and the transmembrane domain; and a C-terminal truncation that results in deletion of the cytoplasmic domain, wherein the amino acid positions are numbered with reference to the HXB2 numbering scheme.

16. The modified Env polypeptide of claim 15(ii), wherein the CD5 signal peptide sequence comprises residues 1-24 of SEQ ID NO: 3.

17. The modified Env polypeptide of claim 15(ii) or claim 16, wherein the plurality of amino acid substitutions comprises:

(a) an aspartic acid substitution at position 47 ;

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(c) a lysine substitution at position 65;

(d) a threonine substitution at position 106;

(e) an arginine substitution at position 166;

(f) a glutamine substitution at position 170;

(g) a tyrosine substitution at position 302;

(h) a methionine substitution at position 320;

(i) an arginine substitution at position 429;

(j) a glutamine substitution at position 432;

(k) an arginine substitution at position 500;

(l) a tyrosine substitution at position 513;

(m) an arginine substitution at position 519;

(n) an arginine substitution at position 520;

(o) a glycine substitution at position 568;

(p) a glycine substitution at position 636; or

(q) a combination of two or more of (a) to (p), wherein the amino acid position is numbered with reference to the HXB2 numbering scheme.

18. The modified Env polypeptide of claim 17, comprising the combination of (a) to (o).

19. The modified Env polypeptide of any one of claim 15(ii) and claims 16-18, wherein the first heterologous peptide linker comprises the amino acid sequence G4SG4S (SEQ ID NO: 13).

20. The modified Env polypeptide of any one of claim 15(ii) and claims 16-19, wherein the second heterologous peptide linker comprises the amino acid sequence G4SK (SEQ ID NO: 10).

21. The modified Env polypeptide of any one of claim 15(ii) and claims 16-20, wherein the C-terminal truncation results in an Env 145 polypeptide.

22. The modified Env polypeptide of any one of claim 15(ii) and claims 16-21, wherein the amino acid sequence of the Env polypeptide is at least 95% identical to SEQ ID NO: 3.

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23. The modified Env polypeptide of any one of claim 15(ii) and claims 16-22, wherein the amino acid sequence of the Env polypeptide comprises or consists of SEQ ID NO: 3.

24. The modified Env polypeptide of claim 15(i), wherein the cleavage competent sequence comprises residues 487-497 of SEQ ID NO: 6.

25. The modified Env polypeptide of claim 15(i) or claim 24, wherein the C-terminal truncation results in an Env 150 polypeptide.

26. The modified Env polypeptide of claim 15(i), claim 24 or claim 25, wherein the amino acid sequence of the Env polypeptide is at least 95% identical to SEQ ID NO: 6.

27. The modified Env polypeptide of any one of claim 15(i) and claims 24-26, wherein the amino acid sequence of the Env polypeptide comprises or consists of SEQ ID NO: 6.

28. An HIV-1 envelope (Env) trimer comprising the modified Env polypeptide of any one of claims 15-27.

29. A nucleic acid molecule encoding the modified Env polypeptide of any one of claim 15(i) and claims 16-23.

30. The nucleic acid molecule of claim 29, comprising a nucleotide sequence at least 95% identical to SEQ ID NO: 2.

31. The nucleic acid molecule of claim 29 or claim 30, wherein the nucleotide sequence comprises of consists of SEQ ID NO: 2.

32. A vector comprising the nucleic acid molecule of any one of claims 29-31.

33. The vector of claim 32, wherein the vector is an adenovirus vector.

34. The vector of claim 33, wherein the adenovirus vector is a replication competent adenovirus type 4 (Ad4) vector.

35. The vector of claim 34, wherein the Ad4 vector comprises a deletion of the adenovirus E3 region.

36. The vector of any one of claims 32-35, comprising a nucleotide sequence at least 95% identical to SEQ ID NO: 1.

37. The vector of claim 36, wherein the nucleotide sequence comprises or consists of SEQ ID NO: 1.

38. A nucleic acid molecule encoding the modified Env polypeptide of any one of claim 15(ii) and claims 24-27.

39. The nucleic acid molecule of claim 38, comprising a nucleotide sequence at least 95% identical to SEQ ID NO: 5.

40. The nucleic acid molecule of claim 39, wherein the nucleotide sequence comprises or consists of SEQ ID NO: 5.

41. A vector comprising the nucleic acid molecule of any one of claims 38-40.

42. The vector of claim 41, wherein the vector is an adenovirus vector.

43. The vector of claim 42, wherein the adenovirus vector is a replication competent adenovirus type 4 (Ad4) vector.

44. The vector of claim 43, wherein the Ad4 vector comprises a partial deletion of the adenovirus E3 region.

45. The vector of any one of claims 41-44, comprising a nucleotide sequence at least 95% identical to SEQ ID NO: 4.

46. The vector of claim 45, wherein the nucleotide sequence comprises or consists of SEQ ID NO: 4.

47. An immunogenic composition comprising the recombinant adenovirus of any one of claims 1-14, the modified Env polypeptide of any one of claims 15-27, the Env trimer of claim 28, the nucleic acid molecule of any one of claims 29-31 and 38-40, or the vector of any one of claims 32-37 and 41-46, and a pharmaceutically acceptable carrier.

48. The immunogenic composition of claim 47 further comprising an adjuvant.

49. A method of eliciting an immune response against HIV-1 in a subject, comprising administering to the subject an effective amount of the recombinant adenovirus of any one of claims 1-14, the modified Env polypeptide of any one of claims 15-27, the Env trimer of claim 28, the nucleic acid molecule of any one of claims 29-31 and 38-40, the vector of any one of claims 32- 37 and 41-46, or the immunogenic composition of claim 47 or claim 48.

50. The method of claim 49, wherein administration comprises intranasal administration or oral administration.

51. The recombinant adenovirus of any one of claims 1-14, the modified Env polypeptide of any one of claims 15-27, the Env trimer of claim 28, the nucleic acid molecule of any one of claims 29-31 and 38-40, the vector of any one of claims 32-37 and 41-46, or the immunogenic composition of claim 47 or claim 48, for use in a method of eliciting an immune response against HIV-1 in a subject.

52. Use of the recombinant adenovirus of any one of claims 1-14, the modified Env polypeptide of any one of claims 15-27, the Env trimer of claim 28, the nucleic acid molecule of any one of claims 29-31 and 38-40, the vector of any one of claims 32-37 and 41-46, or the immunogenic composition of claim 47 or claim 48, for eliciting an immune response against HIV-1 in a subject.

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