WO2011047031A2 - Eliciting immune responses using recombinant mva viruses expressing hiv env, gag and pol anitgens - Google Patents

Abstract

Methods for eliciting beneficial immune responses against HIV by administering to a subject a recombinant MVA virus expressing HIV env, gag, and pol antigens are described. The recombinant MVA is administered at least three times and, in certain embodiments, is administered to a patient that has not been treated with a DNA vaccine directed against HIV (i.e., has not been treated with a nucleic acid molecule encoding one or more HIV antigens). The methods can elicit production IgA antibodies directed against HIV in rectal secretions of a treated subject.

Description

ELICITING IMMUNE RESPONSES USING RECOMBINANT MVA VIRUSES EXPRESSING HTV ENV, GAG AND POL ANITGENS

Field of the Invention

The invention provides methods for eliciting immune responses by administering a modified vaccinia Ankara (MVA) expressing human immunodeficiency virus (HIV) env, gag, and pol antigens.

Description of the Related Art

DNA vectors and MVA expressing HIV antigens have been used to elicit immune responses in patients. In many cases, administration of a DNA vector is used to prime the immune response and subsequent administration of MVA is used to boost the immune response.

Summary of the Invention

Described herein are methods for eliciting beneficial immune responses against HIV by administering to a subject a recombinant MVA virus expressing HIV env, gag, and pol antigens. The recombinant MVA is administered at least three times and, in certain embodiments, is administered to a patient that has not been treated with a DNA vaccine directed against HIV (i.e., has not been treated with a nucleic acid molecule encoding one or more HIV antigens). The recombinant MVA can encode either a full-length HIV Env protein composed of both gpl20 and gp41 subunits or the recombinant MVA can encode gpl20 and a truncated gp41. For example, a truncated gp41 subunit can include the membrane spanning domain and the ectodomain of gp41, but lackspart or all of the cytoplasmic domain of gp41 (e.g., lacksthe carboxy-terminal 112, 113, 1 14, 115, 116, 1 17 or 118 amino acids of gp41). The HTV gag and pol can include one or more mutations as described in greater detail below.

In the methods described herein, the recombinant MVA serves to both prime and boost immune responses. The recombinant MVA is administered three times in a so-called MMM protocol or more than three times (e.g., 4 or 5 times). Preclinical testing administration of a recombinant MVA expressing gag, pol and env antigens in an MMM protocol exhibited two advantages over a protocol entailing priming twice with a DNA vector and boosting twice with a recombinant MVA (DDMM protocol): (i) higher induction of anti -viral IgA in rectal secretions and (ii) induction of higher avidity IgG to the native from of Env. In clinical testing, administration of recombinant MVA expressing gag, pol and env antigens in an MMM protocol also elicited higher titer Ab responses than the DDMM regimen. These results are surprising given that the recombinant MVA in the DDMM protocol was identical to the recombinant MVA in the MMM protocol and suggests that MVA priming and boosting may raise a more favorable antibody response for preventing an infection and that this may be particularly true for mucosal infections.

Suitable recombinant MVA for use in the methods described herein include: MVA 65 A/G, MVA 62B, and MVA 71C, all of which are described in US 2008/0193483, incorporated herein by reference.

In various embodiments, the MVA includes a HIV env encoding sequcne and an HIV gag/pol encoding seuquence. The HTV env, gag, or pol encoding sequence is taken from circulating recombinant form AG and the HIV env encoding sequence or modified encoding sequence thereof has SEQ ID NO: 1 or a sequence having at least about 85%, 90%, 95%, 97%, 98%), 99% or 99.9% identity thereto, and the HIV gag and pol encoding sequence or modified encoding sequence has SEQ ID NO: 2 or a sequence having at least about 85%, 90%, 95%, 97%), 98%), 99% or 99.9% identity thereto; or the HIV env, gag, or pol encoding sequence or modified encoding sequence thereof is taken from clade B and the HIV env encoding sequence or modified encoding sequence has SEQ ID NO: 3 or a sequence having at least about 85%, 90%, 95%, 97%), 98%, 99% or 99.9% identity thereto, and the HIV gag and pol encoding sequence or modified encoding sequence has SEQ ID NO: 4 or a sequence having at least about 85%, 90%), 95%, 97%, 98%, 99% or 99.9% identity thereto; or the HTV env, gag, or pol encoding sequence or modified encoding sequence is taken from clade C and the HIV env encoding sequence or modified encoding sequence has SEQ ID NO: 5 or a sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 99.9% identity thereto, and the HTV gag and pol encoding sequence (s) or modified encoding sequence (s) thereof has SEQ ID NO: 6 or a sequence having at least about 85%, 90%, 95%, 97%, 98%, 99% or 99.9% identity thereto.

In various embodiments, the MVA encodes an Env that is at least 80%>. 85%. 90%, 95%, or 98% identical to the Env encoded by SEQ ID NO:l, 3 or 5 and the MVA encodes a Gag/Pol that is at least about least 80%. 85%. 90%, 95%, or 98% identical to the Gag/Pol encoded by SEQ ID NO:2, 4 or 6. The MVA virus used to make the recombinant MVA 65A/G is preferably MVA 1974/NIH Clone 1. Preferably the env and the gag/pol encoding sequences are inserted into different locations in the MVA genome, for example, the env encoding sequence is inserted into deletion site Π of MVA genome and the A/G gag/pol encoding sequence is inserted into deletion site ΠΙ of MVA genome. In various embodiments the transcription of the sequence encoding the HIV Env antigen is under the control of a first promoter and transcription of the sequence encoding the HIV Gag/Pol antigens is under the control of a second promoter and the non-coding sequence between the first promoter and the initiation codon of the sequence encoding the HIV Env antigen comprises the sequence ATG or other initiation codon sequence (for example GTG or TTG). In certain embodiements the ATG or other intiation codon sequence is not in frame with the initiation codon of the sequence encoding the HIV Env antigen.

In various embodiments, the MVA encodes an Env that is at least least 80%. 85%. 90%, 95%, or 98% identical to the Env encoded by SEQ ID NO:7, 8, 9 or 10. In other embodiments the MVA encodes an Env that is at least least 80%. 85%. 90%, 95%, or 98% identical to an Env described in the Los Alamos HIV Sequence Database. For example, the Env of HXB2 (GenBank Accession K03455). The HXB2 Env sequence is in SEQ ID NO: 11). The recombinant MVA can encode either a full-length HIV Env protein composed of both gpl20 and gp41 subunits or the recombinant MVA can encode gpl20 and a truncated gp41. For example, gp41 subunit in some cases is comprised of the membrane spanning domain and the ectodomain, but may lack part or all of the cytoplasmic domain of gp41 (e.g., lacking the carboxy-terminal 112, 1 13, 114, 115, 1 16, 117 or 1 18 amino acids of gp41or the carboxy- terminal 1 12, 1 13, 114, 1 15, 116, 1 17 or 118 amino acids of a full-length Env sequence such as those in the Los Alamos database, e.g., SEQ ID NO: l 1).

In various embodiments, the MVA encodes an Gag that is at least least 80%. 85%. 90%, 95%, or 98% identical to the Env encoded by SEQ ID NO: 12, 13, or 14. In other embodiments the MVA encodes an Env that is at least least 80%. 85%. 90%, 95%, or 98% identical to an Gag described in the Los Alamos HIV Sequence Database. For example, the Gag of HXB2 (GenBank Accession K03455). The HXB2 Gag sequence is in SEQ ID NO: 15).

In various embodiments, the MVA encodes a Pol that is at least least 80%. 85%. 90%, 95%, or 98% identical to the Env encoded by SEQ ID NO: 16, 17, or 18. In other embodiments the MVA encodes an Env that is at least least 80%. 85%. 90%, 95%, or 98% identical to an Gag described in the Los Alamos HIV Sequence Database. For example, the Pol of HXB2 (GenBank Accession K03455). The Pol antigen can include fewer than all of the amino acids of p31 integrase. Thus, in some cases it includes 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 50, 100, 150, 200 of the amino terminal amino acids of p31 integrase.

FIGURES

Figure 1. Ml 1 macaque trial: MMM immunizations elicit higher avidity anti-Env Ab than DDMM immunizations. Symbols indicate individual macaques. Note how the MMM immunizations elicit higher avidity anti-Env Ab than the DDMM immunizations even after the 2^nd MVA delivery (MM). Avidity was measured against full length SIV239 Env that had been conA-captured from Triton-X-100 dissociated VLPs . Avidity index is the % of bound Ab that resists a 1.5M NaSCN wash[l][39].

Figure 2. SIV Gag and Env specific IFNy -expressing T cell responses post vaccination. Responding T cells were tested for using intracellular cytokine staining following stimulation with pools of SIVmac239 Gag and Env peptides (15mers overlapping by 1 1). Note how CD4 responses are 5 to 10-fold higher in the DDMM than in the MMM group, whereas the magnitudes of the CD8 responses are similar in the two groups. Responses below 0.01% of total CD4 or CD8 T cells are considered below the limit of detection

Figure 3. Kaplan-Meier plot for % of DDMM or MMM vaccinated animals protected against a repeated intrarectal challenge with a heterologous SIV. Animals were vaccinated with SrVmac239 and challenged with 5000 TCID₅₀ of SIVsmE660. This dose of virus resulted in 30% of the unvaccinated animasl (black dashed line) becoming infected at each challenge. Vaccinated groups are indicated at the top of the schematic and data for vaccinated animals are presented as solid colored lines (green for DDMM and magenta for MMM).

Figure 4. Temporal levels of post challenge virus in plasma of vaccinated and challenged animals that became infected. Post challenge data have been synchronized by plotting the first week that infections was detected as week 1. Each line presents data for an individual animal. Black, unvaccinated macaques; green , DDMM vaccinated macaques;

magenta, MMM vaccinated macaques. The lower limit for detection of viral RNA was 200 copies per ml of plasma, ented to Figure 5. Schematic representations of the HIV-1 vaccines and study design. (A) Schematics for DNA and recombinant MVA immunogens. (B) HVTN 065 trial schema.

CMVIE, CMV immediate early promoter; gag, HIV-1 gene encoding group specific antigens; PR and RT, protease and reverse transcriptase encoding regions of HIV-1 pol; tat, vpu, and rev, HIV-1 regulatory genes; gpl20 and gp41, surface and transmembrane subunit-encoding regions of HIV-1 env ; gp41tr, gp41 with a 1 15 amino acid C-terminal truncation; BGHpA, bovine growth hormone polyadenylation sequence; x, presence of inactivating point mutations in packaging sequences for viral R A in Gag and the protease, reverse transcriptase, strand transfer and RNase H activities of Pol [24]; PmH5, the modified H5 early/late vaccinia promoter; deletions II and III, naturally occurring deletions in MVA.

Figure 6. Reactogenicity of study vaccine regimens. The percent of participants with local pain and/or tenderness (A) or any systemic symptom (B) following each vaccine dose is shown. Reactions were graded as none, mild, moderate, or severe. The vaccine groups are given at the top of the schematics and the immunization status of groups at the bottom. D, DNA; M, MVA, P, placebo. The number of Ds and Ms indicate the number of immunizations, for example, DDM means two DNA and one MVA immunization. For more detail, see Materials and Methods.

Figure 7. Immune response rates determined in end point assays. Response rates for CD4+T cells (A), CD8+T cells (B) and Anti-Env Ab (C). Responses for CD4+and CD8+T cells are for responses to Gag, Env, or Pol measured as IFN-γ or IL-2 producing cells scored using intracellular cytokine staining (ICS) following stimulation with potential T cell epitope peptide pools. Response rate for anti-Env Ab were measured using an ELISA for the sp400 peptide, a peptide representing the immunodominant region of gp41. Lymphocytes and sera for determining response rates were harvested at 2 weeks following immunizations. Significant differences between groups are indicated where appropriate. All assays were performed in HVTN laboratories on frozen samples. Letters at the bottom of schematics indicate group, and the immunization status of groups (see legend to Fig. 2 for designations). See Materials and Methods for more detail.

Figure 8. Magnitude, persistence and polyfunctionality of vaccine-induced T cell responses. In panels A and B, the magnitudes of CD4+(A) and CD8+(B) T cell responses following full dose DDMM and MMM vaccine regimens are shown . Data represent responses directed against Gag and Env as measured by IFN-γ and/or IL-2 production of CD4+ and CD8+ T cells in an ICS assay (for more detail see legend to Figure 7 and Materials and Methods). Boxplots represent median and 25^th and 75^th percentiles for positive data (indicated by red points); blue points indicate negative data. Response rates are shown below schematics as number of participants positive out of number of participants tested, with the % responders given immediately below. Pre&P, prebleed at baseline and placebos, +2wk, samples harvested at 2 weeks post an injection; +3mo and + 6mo; samples harvested at 3 or 6 months post the last injection. Letters at the bottom of schematics indicate group, and the immunization status of groups (see legend to Fig. 2 for designations). *, p<0.05 for CD4 T cell response frequency when compared to the +2 wk time point following the final vaccination in the DDMM regimen. **, p<0.05 for CD8 T cell responses frequency when compared to that seen after the first MVA boost in the DDMM regimen. (C-F) Polyfunctionality of the positive responses for IFN-γ, IL-2, and TNF-a production measured using multicolor flow cytometry and Boolean analyses. Panels C and D show the percentages of CD4+ (C) and CD8+ (D) T cells producing single cytokines. Panels E and F show the degree of polyfuntionality for the CD4+ (E) and CD8+ (F) responses where 1 indicates the % of responding cells producing a single cytokine; 2, the % producing two cytokines; and 3, the % producing three cytokines.

Figure 9. Breadth/depth and magnitude of T cell responses to Gag and Env. (A) % responders, median magnitudes and total # of recognized peptide pools for the 4 vaccine regimens. The total # of recognized pools represents the sum of all of the peptide pools recognized in assays successfully completed for a group normalized to the maximum number of individuals tested for CD4 and CD8 T cell responses in that group. Note that this normalization was largest for the low dose DDMM group that had only 10 participants compared with the 30 participants in the other groups. (B and C) % of responders with CD4+ (B) or CD8+ (C) T cells recognizing different numbers of peptide pools . The numbers in the graph are the median number of peptide pools recognized by responders to a particular regimen. See legend to Figure 5 for interpretation of designations. Note that the numbers represent the breadth and/or depth of induced T cells as previously defined [34]. Potential T cell epitope pools are grouped depending on the frequency of HIV- 1 epitope variants, so variants of the same epitope may be in different peptide pools [32]. Figure 10. Magnitudes and response rates of Env binding and neutralizing antibodies. (A) Binding Ab for sp400, a peptide representing the immunodominant region of gp41. (B) Binding Ab for ADA gpl20. (C) Neutralizing Ab for HTV-IMN- Designations below schematics indicate groups and response rates (see legend to Fig. 4 for details). The boxplots show median and 25th and 75th percentiles for positive data (indicated by red points). Blue points indicate negative data. Data for determining P values include only positive data (see legend to Fig. 4 for more information). (D) Percent of positive MN neutralization responses also neutralizing other tier 1 isolates. Seventeen of the samples demonstrating neutralization against HIV- IMN were evaluated further. The tier 1 isolates are shown including HIV-1SFI62, HIV-1W₆ID (T-cell lab-adapted strain), and HIV-I BAL-

Figure 11. Vector-specific T cells. The percent of MVA-specific CD4+ or CD8+ T cells producing IFN-γ by ICS assay is shown for samples analyzed at 1 week following the first or second MVA boost for the DDMM and MMM vaccine regimens. The percent of participants with vector-specific T cell responses (response rate) is given below the schematic.

Figure 12. Various sequences encoding Env antigens and Gag/Pol antigens as well as various antigen sequences.

Detailed Description

Brief Overview of Preclinical Study Results Conducted in Macaques

In preclinical testing administration of recombinant MVA expressing gag, pol and env in an MMM protocol exhibited two advantages over a protocol entailing priming twice with a DNA vector and boosting twice with a recombinant MVA (DDMM protocol): (i) induction of anti -viral IgA in rectal secretions and (ii) induction of high avidity IgG to the native from of Env.

Colorectal Secretions

In a study conducted in macaques (the "Ml 1 Trial"), the MMM regimen elicited a much higher frequency of antiviral IgA in colorectal secretions than the DDMM regimen (Table 1). Whereas anti -viral mucosal IgA was only occasionally seen in the rectal secretions of DDMM vaccinated animals, it was present, at least transiently, in the majority of MMM- vaccinated macaques. The mucosal IgA responses in the MMM vaccinated animals detected both Gag-Pol, and Env. Importantly, in two prior preclinical studies, the presence of anti-viral IgA in rectal secretions has correlated with increased protection[l, 2]. In the 2^nd of these studies [2] , the presence of antiviral IgA in rectal secretions was associated with reduced titers of viral RNA at the colorectal challenge site and a reduced tempo of infection.

Table 1 : IgA antibodies in rectal secretions of trial M1 1 macaques

IgA assays were conducted in the laboratory of Dr. Pamela Kozlowski, LSU. Highlighted values are significant. To be significant, the specific activity for SIV env or gag.pol antigens had to be more than or equal to 0.145 or 0.224, respectively (the mean specific activity + 3 sd for naive macaques) and 2.7-fold more than preimmune specific activity.

To facilitate calculations, samples with no detectable antibody were assigned specific activity values corresponding to the mean of negative controls (0.049 for env; 0.083 for gag.pol).

The elicitation of anti-viral IgA has been a much sought phenomenon for HIV vaccines, because most infections worldwide are through mucosal surfaces. The first condition that we found to elicit mucosal IgA was the use of GM-CSF as an adjuvant for the DNA prime. GM- CSF has recently been shown to stimulate dendritic cells to produce retinoic acid[3], which in turn stimulates mucosal trafficking and IgA production[3-5]. The two other trial regimens that we have found associated with mucosal IgA are DDMM immunizations in vaccinia preimmune animals[2] and MMM immunizations (Ml 1 vaccine trials). Because of the merit of mucosal IgA for an HIV/ AIDS vaccine, we consider that induction of mucosal IgA by MMM immunizations warrants pursuit in clinical as well as preclinical settings. Avidity of the anti-Env Ab response

In preclinical trials, the avidity (tightness of binding) of the anti-Env Ab response correlated with reduced levels of peak viremia. Indeed, an inverse correlation between avidity for full length Env and peak viremia has been observed in all of our preclinical trials in which it has been measured[l, 2, 6]. In the different trials, the extent of the reduction of the peak viremia has reflected the relationship of the Env used as immunogen and the Env present in the challenge virus(Table 2). For the correlation to be seen, avidity had to be measured against the native form of Env (conA captured from triton-X-100 disrupted VLPs or pseudo virions). The avidity indices for gpl20 or gpl40 subunits of Env have not necessarily measured responses against epitopes that correlate with protection[l , 6].

In the Ml 1 Trial, the avidity index of the anti-Env Ab elicited by MMM immunizations (median of 58) is twice that of the avidity of the anti Env IgG elicited by DDMM

immunization (median of 30) (Figure 1). This is a large avidity differential.

Table 2. Summary of Associations of Avidity with Protection

Avidity is measured in duplicate ELISAs, one subjected to a 1.5 M NaSCN wash, and one to a PBS

wash. The avidity index is the dilution at which the NaSCN washed sample has an OD of 0.5

divided by the dilution at which the PBS washed sample has an OD of 0.5 x 100. The recombinant MVA used in the methods described herein, expresses a native, transmembrane bound Env (full-length except for deletion of all or a portion of the cytoplamsic domain of gp41). Without being bound by any particular theory, it may be that Ab elicited by native Env, can recognize Env on virions and infected cells and that if this Ab binds tightly enough, it can initiate Fc-mediated mechanisms of protection such as C -mediated lysis, opsonization, antibody dependent cellular cytotoxicity (ADCC), and antibody dependent cell- mediated virus inhibition (ADCVI). Avidity also has a feature that is desirable for an

HTV/AIDS vaccine: when elicited by a native trimeric membrane bound Env, it has breadth for incident isolates[6]. This breadth does not extend across clades, but does include incident isolates within a clade. The breadth in the avidity of the present recombinant MVA-elicited sera for Envs of incident isolates is consistent with the good breadth for patient isolates found for C'-mediated lysis[7, 8], ADCC[9-11] and ADCVI[12] activities in patient sera.

Prior examples of vaccines for which the avidity of an Ab response has been found to be important for protection include the conjugate vaccines. These vaccines convert T-cell independent to T-cell-dependent immunogens and allow Ab stimulated by polysaccharides to undergo affinity maturation in children under two years of age. For example, the avidity of the Ab responses elicited by Haemophilus influenzae type B (Hib)[13] and Streptococcus pneumononiae (pneumococcus)[14] are key to their protective activities. Thus, it is likely that Ab avidity is important in vaccine-mediated protection. The measurement of avidity for HIV-1 immunogens may be of particular import because of the slow maturation of Ab to the highly glycosylated Env[15]. For many other viral targets, however, avidity maturation is sufficiently rapid that it does not merit measurement.

Both the DDMM and MMM regimens elicited both CD4+ and CD 8+ T cell responses (Figure 2). The magnitudes of elicited CD8+ T cells were similar in both regimens, whereas the magnitude of CD4+ responses was higher in the DDMM regimen. Thus the MMM regimen had excellent ability to elicit both T cells and antibody specific for the HIV vaccine insert.

Preclinical protection studies.

Six months following the final immunization, macaques in the Ml 1 trial were subjected to a repeat heterologous intrarectal STV challenge [16] [17] [18]. The vaccine in the Ml 1 trial was a SrVmac239 vaccine, whereas the heterologous challenge was SIVsmE660. The genetic distance between SrVmac239 and SrVsmE660 is 83% in Env and 91% in Gag, a difference that is similar to the gtenetic difference between circulating HTV-1 isolates within a clade[19]. An intrarectal challenge of 5000 tissue culture infectious doses₅₀ was adminstered weekly for 6 weeks following which there was a 3 week pause and then a second series of 6 weekly challenges. The results of this challenge revealed the simpler MMM regimen showing a trend for better protection than the more complex DDMM regimen (Figure 3). Following the first 6 challenges only 3/8 MMM vaccinated animals were infected whereas 5/8 DDMM vaccinated animals were infected and 8/9 unvaccinated controls had become infected. By 12 challenges all of the unvaccinated animals were infected and 2/8 DDMM and MMM vaccinated animals remained uninfected. Analysis of the rate of infection in the unvaccinated controls revgealed that the challenge had infected 30% of the macaques at each dose. This transmission per exposure is 100 to 1000-times higher than in typical heterosexual

transmission and 10 to 1000 times higher than in typical homosexual transmission[20]. Thus there is considerable expectation that this regimen with a human vaccine might provide quite good protection against a mucosal challenge.

Macaques that became infected were monitored for their levels of post challenge viremia to determine whether the vaccines had controlled infections (Figure 4). For these analyses, infections were "synchronized" by assigning week 1 of infection to the 1^st week that the infection had been detected. These analyses did not reveal statistically different control between the unvaccinated, DDMM vaccinated and MMM vaccinated animals within our group sizes. However the data were consistent with the MMM animals having a trend towards the lowest peak viremia and the best control at set point. Thus the MMM vaccine showed good potential for both prevention of infection and control of infection in the macaques that became infected

Preclinical experience with protein boosts of DDD, DDMM and MMM immunizations.

Comparison of the titers of anti-Env Ab responses elicited in our preclinical macaque studies and in our clinical studies in humans (see below) reveal our vaccines eliciting 10 to 50 times higher titers of binding antibody for Env in macaques than humans. Since Ab responses are a likely correlate for prevention of infection, a protein boost of a MMM immunization may be important for achieving needed titers of protective antibody. Our DNA and MVA immunogens express native, membrane-bound trimeric forms of Env. Prior trials using protein boosts of our DNA and MVA vaccines have utilized recombinant gpl20 (a monomer of the CD4 binding subunit of Env) administered with an alum adjuvant[21], oligomeric gpl40 (soluble oligomers of the extracellular domain of gp41 and gpl20) administered with an QS21 aduvant [22] and gpl60 (a detergent solubilized full length form of Env) administered with incomplete Freunds adjuvant[23]. These boosts have increased the titers of elicited Ab by 10 to 50-fold. When tested against high dose challenges that infect every single macaque at the first dose, these boosts have not prevented infection and have had overall limited effects on post challenge control of infection. However, when used to boost a MMM primed response against a repeated more moderate dose challenge, these boosts might increase the levels of antibody that are contributing to the prevention of infection. This is likely because, a protein boost of the MVA-primed response would preferentially boost shared epitopes between the native forms of Env in our immunogens and the boosting protein; whereas it would not boost displayed in the subunit protein boosts which are hidden in the. native trimers or are unique to partially denatured gpl20, gpl40 or gpl60 proteins B cell epitopes that would be presented to the immune system for the first time by the recombinant protein. Thus a recombinant protein vaccine may raise much more effective Ab by boosting a response that was primed by an MVA vaccine expressing native trimeric membrane-bound Env than by a response that was primed and boosted using the recombinant protein and the higher titers of protein elicited by this boost may be important for preventing infection.

Summary of Clinical Studies

Recombinant DNA and modified vaccinia Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. The HVTN-065 is Phase 1 clinical trial was undertaken to compare the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine priming. Intramuscular needle injections were used to deliver placebo (P), two doses of DNA followed by two doses of rMVA (DDMM), one dose of DNA followed by two doses of rMVA (DMM), or three doses of rMVA (MMM) to HIY

seronegative participants. Vaccines

The GeoVax HIV-1 DNA vaccine, pGA2/JS7 DNA (JS7), produces non-infectious virus-like particles (VLPs), and encodes HIV-1HXB-2 Gag, HIV-le_HioProtease (PR) and Reverse Transcriptase (RT), and Env, Tat, Rev, and Vpu derived from a recombinant of the HXB-2 and ADA strains of HIV- 1 (Figure 5 A). The vaccine is rendered non-infectious by gene deletions and inactivating point mutations [24] [25].

Modified Vaccinia Ankara MVA/HIV62 (MVA62) encodes HIV-1 Gag, PR, RT and Env from the same sequences as JS7 and also produces non-infectious VLP (fFgurelA)

[26] [27]. MVA62 contains the RT but not the Gag and PR mutations of JS7. The ADA Env gene is truncated by 115 C-terminal amino acids of gp41 resulting in higher surface expression of Env and the elicitation of higher Ab responses in mice [28].

Study design

HIV Vaccine Trials Network (HVTN) protocol 065 was a randomized, double blind, placebo controlled trial conducted at six clinical sites in the United States (Figure IB). Adults aged 18-49 years who were deemed healthy based on medical history, physical exam, laboratory tests, troponin levels, and electrocardiogram (EKG) were enrolled. The study was designed with 10 participants receiving 0.3 mg of the JS7 and 10⁷ tissue culture infective dose (TCID₅₀) of MVA62 (1/10^th dose) at 8 week intervals in the DDMM schedule. After a safety review of the 1/10^th dose DNA vaccinations, 30 participants were randomized to receive full doses of the vaccines (3 mg and 10^s TCID₅₀, respectively) in the DDMM sequence. Once the full dose DDMM regimen was demonstrated to be adequately safe and immunogenic, part B of the trial was started. This included the enrollment of 30 participants to receive full dose vaccines in the DMM or MMM sequences with immunizations administered at 0, 8 and 24 weeks. The placebo product used for all groups was saline and placebo participants were enrolled at the ratio of 1 :5, placebo recipients :vaccinees.

Vaccines were delivered as 1 ml into the deltoid region intramuscularly by needle injection. Safety evaluations included physical examinations, standard clinical chemistry and hematological tests supplemented with tests to expand the ability to identify potential cardiac issues, which included troponin levels and 12-lead electrocardiograms (EKG). Local injection site (pain, tenderness, redness, erythema, and induration) and systemic (malaise, headache, fever, chills, myalgias, arthralgias, nausea, vomiting, and fatique) reactogenicity symptoms were assessed for three days following each vaccination or until resolution. Reactions were graded as mild, moderate, or severe according to standard criteria (http://rcc.tech- res . com/ safety andpharmaco vigilance/) .

Immune response assays

T cell responses. Peripheral blood mononuclear cells (PBMC) cryopreserved within 8 hours of venipuncture were obtained two weeks after each vaccination and at three and six months after the last vaccination [29]. HIV-specific T cells responses were measured using intracellular cytokine staining (ICS) conducted at HVTN central laboratories [30];[31]; .

Global potential T cell epitope [32]peptide pools representing HIV Env (3 pools), Gag (2 pools), and Pol (3 pools) were used at the final concentration of 1 μg for each peptide per ml. Cells were first stained with the Violet Live/Dead Fixable Dead Cell Stain [33] then fixed, permeabilized, and stained with the following reagents: CD3-ECD, CD4-FITC, CD8-PerCP- Cy5.5, IFN-y-PE-Cy7, IL-2-PE, TNF-a-Alexa 700 and IL-4-APC. Positive responses were identified using one-sided Fisher's exact test to support comparison of differences between background measurements and the numbers of CD4+ or CD8+ T cells producing IFN-γ and/or IL-2 in response to peptide stimulation [31]. The breadth and/or depth of T cell responses [34] was calculated by the number of peptide pools eliciting a positive reaction per vaccinee (8 maximum).

Assays to measure MVA-specific T cell responses were conducted at the Emory Vaccine Center using similar methods. For the MVA assay, stimulations were conducted by infecting PBMC with Western Reserve vaccinia virus at a multiplicity of infection of 1-2 for six hours following which Golgi Plug (Pharmigen) was added and incubations were continued at 37 °C overnight. Antibody reagents used were anti-CD3 Alexa 488, anti-IL-2-PE, anti-IFN- γ APC, anti-CD4 PerCP or anti-CD8 PerCP. Positive results were defined as twice the background of unstimulated cells and >0.01% of the total CD4+ or CD8+ T cells.

Antibodies. Standard HIV ELISA and Western blot testing (Abbott Labs) was performed in participants following the final vaccination. Analyses for Env binding and neutralizing activity were conducted by the HVTN laboratories. An enzyme-linked immunosorbent assays (ELISA) based on alkaline phosphatase and the AttoPhos fluorescent substrate (Hoffman La Roche) was used to measure total binding Ab to the HIV gp41 immunodominant peptide, SP400, (RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKI). Fluorescent readings were measured using a M2 plate reader (Molecular Devices, Sunnyvale, CA) and mean fluorescent intensity for each pair of replicates, with the background subtracted, was calculated. Standard curves were generated from the plot of fluorescence against the log of serum dilution and sigmoidal curves were fit using a four-parameter logistic equation (Softmax Pro). Positive responses for each serum dilution were defined as three times the value at baseline.

HIV neutralization was measured as a reduction in luciferase reporter gene expression after a single round of infection in TZM-bl cells. Neutralization titers were defined as the dilution at which relative luminescence units (RLU) were reduced by 50% compared to virus control wells after subtraction of background. An assay stock of HIV- 1 MN was produced in H9 cells and a stock of molecularly cloned ADA Env-pseudotyped virus by transfection in 293T cells. Samples were considered positive if the neutralization titer that reduced cell killing by 50% was > 25. Ancillary ELISAs were completed at GeoVax to determine titers of Env Ab specific for the monomelic ADA gpl20 produced using a recombinant vaccinia virus.

Microtiter plates were coated with sheep Ab to the C terminus of gpl20 (D7324, Aalto BioReagent Ltd, Dublin), ADA gpl20 was captured and serial dilutions of human sera were incubated on duplicate wells with or without ADA gpl20. Serial dilutions of HIV-Ig (3957, NIH AIDS Research and Reference Reagent Program) with known levels of g l20 binding Ab were used as the standard on each plate. Bound antibody was detected with IgG-specific antisera conjugated to peroxidase and TMB peroxidase substrate (KPL, Gaithersburg, MD). Optical densities were read using a Molecular Devices machine and the ng of bound antibody estimated from the HIV-Ig sigmoidal curve generated using four parameter logistic software (Softmax Pro). Samples were considered positive if they were at least 3 times background and had a total estimated concentration of >10 ng of anti-gpl20 Ab per ml.

Statistical Analysis For safety, the number and percentage of participants

experiencing each type of reactogenicity sign or symptom were tabulated by severity and vaccine regimen using MedDRA preferred terms. Then for a given sign or symptom, each participant's reactogenicity was counted once under the maximum severity for all injection visits or the strongest recorded causal relationship to treatment. For immunogenicity, boxplots of local laboratory values by treatment were generated for baseline values and for values measured during the course of the study. Comparisons of immune responses between groups used the Wilcoxon rank-sum test and SAS, S-Plus, or R statistical software.

Participant Accrual, Demographic Data, and Vaccine Safety The median age of participants was 24 years and 58% were female. The majority were white (73%) or African- American (16%). All 120 participants received their initial vaccine and 104 (87%) received all prescribed doses. Of those who did not, 8 missed the window period for vaccination or were unable to be contacted, 2 became pregnant, 3 refused to continue injections, one was discontinued from vaccination for a pre-existing condition, and 2 were discontinued from vaccination due to adverse events. One developed chest tightness and dyspnea 30 minutes after vaccination, which was probably related to vaccination; another had an AE that was not related to vaccination.

The vaccines were safe and well tolerated at both doses and using all schedules without severe reactogenicity (Figure 6). Participants had similar mild or no local side effects after placebo and JS7 DNA administrations (at 1/10th or full dose). The low dose MVA62 vaccine was also associated with only mild local side effects. However, the full dose MVA was associated with an increased number of participants experiencing either mild or moderate local reactogenicity (Figure 6A). Most of the local side effects included pain at the injection site. The majority of participants had either no, or mild, systemic side effects with a few moderate reactions, and there were no differences when compared to placebo recipients. There were seven adverse events that were at least probably or definitely attributed to the vaccine and six of these were mild local reactions. One individual experienced a moderate decrease in neutrophils 14 days following the first DNA vaccination, but this resolved and did not recur - following subsequent injections. There were no laboratory abnormalities or EKG changes that could be attributable to this vaccine administration.

Immunogenicity

HIV-1 specific T cell responses. HIV-1 specific T cell responses were readily detected in all groups; however, the response rates depended on the vaccine regimen (Figure 7). The DNA prime enhanced both CD4 and CD8 response rates with two DNA primes (either 1/10^th or full dose) being more effective than a single full dose DNA prime. Vaccine-induced CD4+T cells were measured in 88%» of individuals vaccinated with the 1/10* dose and 77% of those vaccinated with the full dose DDMM regimen. This compared with peak CD4+ response rates of 50% for DMM and 43% for MMM regimens (Figure 7A). Peak CD8+ T cell responses were 33% in the participants receiving 1/10^th dose and 42% in participants receiving the full dose DDMM regimens compared with 22% and 17% in participants in the DMM and MMM regimens, respectively (Figure 7B). The magnitudes of responses were overlapping for all groups with medians between 0.07 to 0.17% of total CD4+T cells and between 0.06 and 0.65% of total CD8+T cells (Figures 8, 9A). Male and female participants had similar response rates (data not shown). The time courses and persistence of T cell responses differed for the full dose DDMM and MMM regimens (Figures 7 and 8). Both the rates and magnitudes of CD4+T cell responses were maximal and remained maximal after the first MVA inoculation in the DDMM and DMM groups whereas responses peaked and then fell after the second dose of MVA62 in the MMM group. In contrast, CD8+T cell response rates, but not magnitudes, increased with the last dose of MVA in the DDMM and DMM groups, whereas these fell slightly with the last dose of MVA62 in the MMM regimen. At six months following the final vaccination, CD4+T cell response rates were 38% for DDMM (49% of their 2-week peak) compared to 8% for MMM (19% of their 2-week peak) (p=0.03); and CD8+T cells at 38% for DDMM (90% of their peak) compared to 4% for MMM (24% of their peak) (Figure 8).

For the DDMM and MMM regimens, the functionality of T cell responses, as measured by co-production of IFN-γ, IL-2 and TNF-a, were similar except for different patterns for single cytokine producing cells (Figure 8 C and D). Among the single cytokine producing CD4+ cells, IL-2 predominated in the DDMM group and TNF-a, in the MMM group. Among single cytokine producing CD8+ T cells, IFN-γ production was most frequent in the DDMM group whereas no single cytokine dominated in the MMM group. For both regimens, approximately one third of the responding cells produced three, two, or one cytokine (Figure 8 E and F). For both regimens, patterns of dual producing CD4+ and CD8+ T cells were similar with the vast majority of dual producing CD8+ T cells producing IFN-γ and TNF-a.

The breadth and depth [34] of T cell responses against two Gag, three Env and three Pol peptide pools revealed responses primarily directed to Gag and Env (Figure 9A). The DNA prime increased the breadth/depth of the T cell response and priming with two doses of DNA (low or full dose) provided a broader response than priming with a single dose of DNA (Figure 9B). Following the final immunization, CD4+ and CD8+ T cells in DDMM recipients recognized medians of 4 and 2 peptide pools, respectively, compared with medians of 1 for CD4+ and CD8+ responses in MMM vaccine recipients. CD4+ T cell responses were evenly distributed between Gag and Env for both DDMM groups, but showed a bias towards Gag in the DMM group and a strong bias towards Gag in the MMM group. The kinetics of T cell responses differed for Gag and Env: following the final MVA dose, CD8+ T cell response rates for Gag increased nine-fold in the DDMM and four-fold in the MMM group; whereas CD8+ responses for Env increased two-fold for DDMM recipients and decreased by three-fold for MMM recipients. The DNA prime biased the response towards CD4+ T cells. This bias was strongest after the 1^st MVA boost when it ranged from a 7 to a 14-fold excess of CD4+ responses over CD8+ responses in the DNA-primed groups compared with a 2.4-fold excess of CD4+ over CD8+ responses in the MMM group.

HIV-1 specific antibody responses. In contrast to T cell responses, HIV-1 specific antibodies were induced more frequently and at higher levels by the full than the 1/10^th dose DDMM regimen and the highest frequencies and titers of Ab responses were induced by the MMM regimen (Figures 7C and 10. After the final vaccine administration, nearly all MMM recipients (96.6%) tested positive by the Abbott HiV-l/HrV-2 ELISA whereas only 73% of the DDMM recipients had seroconverted by this test (p = 0.03). A trend towards an increased number of positive Western blot assays was also seen in the MMM (21.4%) versus the full dose DDMM (4.3%) recipients.

Env-specific antibodies, as measured by binding to the immunodominant SP400 gp41 peptide, binding Ab for a gpl20 monomer of the ADA vaccine Env, and neutralizing activity for HIV-I MN were all highest in the MMM group (Figure 10). Participants receiving the full dose DDMM regimen had the lowest Ab responses, and the DMM group intermediate antibody responses. The higher titers of Ab present in the MMM group were associated with this group receiving three doses of MVA. Following two doses of MVA, Ab responses were overall similar in the DDMM and DMM groups compared to those in the MMM group after two doses of MVA (Figure 10). Samples able to neutralize HIV-IMN were further tested for their ability to neutralize select tier 1 isolates (Figure 10D). While no further neutralization was seen for the DDMM group, recipients of the DMM and MMM regimens were able to neutralize tier 1 isolates with a trend towards the greatest breadth in the MMM group. No neutralization was observed against tier 2 isolates including HIV- 1 ADA- Vector-induced immune responses. The CD4+ and CD8+ T cell responses specific to the MVA vector were assessed following the MVA62 boosts. Subsequent to the first MVA boost, both the response rates and magnitudes of vaccinia-specific CD4+and CD 8+ T cells were significantly lower in those receiving the DNA prime compared to those receiving only MVA62 (pO.0001) (Figure 11).

Consistent with preclinical testing in rhesus macaques [35][36][1];[6], all of the tested regimens induced both T cell and Ab responses. However, the DDMM regimen induced the highest frequency and most persistent CD4+ and CD8+ T cell responses. The DNA prime also increased the breadth/depth of the T cell responses and biased these responses towards CD4+ T cells. On the other hand the MMM regimen induced the highest frequency and magnitude of antibody responses. The single DNA prime (DMM) induced intermediate T cell and Ab responses. The 1/10^th dose DDMM regimen decreased the height of Ab responses but had limited effect on T cell responses.

For all regimens, the number of MVA boosts was important for increasing both anti- Gag CD8+ T cell and anti-Env Ab responses. The last MVA boost increased anti-Gag CD8+ response rates from 4 to 35% and from 4 to 15% for the DDMM and MMM regimens, respectively. The higher Ab responses in the MMM regimen correlated with this regimen receiving 3 MVA inoculations as opposed to the two MVA boosts for the DNA-primed regimens. Prior studies suggest that an additional MVA boost would further increase responses but that this increase would be limited by vaccine-induced immunity curtailing further boosting[l];[37]. Ab responses were also affected by the number of DNA primes, with a single full dose DNA prime tending to give higher Ab responses post the MVA boost than two full dose DNA primes. This could reflect the DNA prime eliciting immune responses that curtailed Ab as well as MVA-specific CD8+ responses. Additionally, the JS7 DNA prime may have elicited a Thl-like response so that it more optimally primed memory T than memory B cells [38]. Recombinant MVA

A suitable MVA for use in constructing recombinant MVA is MVA 1974/ΝΓΗ Clone 1, which was deposited as ATCC Accession No.: PTA-5095 on March 27, 2003 with the

American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA 20110- 2209, USA under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations thereunder (Budapest Treaty).

The recombinant MVA vaccinia viruses can be prepared using widely-known methods. A DNA-construct which contains a DNA-sequence which codes for a foreign polypeptide flanked by MVA DNA sequences adjacent to a naturally occurring deletion, e.g., deletion site III, or other non-essential sites, within the MVA genome, is introduced into cells infected with MVA, to allow homologous recombination. Once the DNA-construct has been introduced into the eukaryotic cell and the foreign DNA has recombined with the viral DNA, it is possible to isolate the desired recombinant vaccinia virus in a manner known per se, preferably with the aid of a marker. The DNA-construct to be inserted can be linear or circular. A plasmid or polymerase chain reaction product is preferred. The DNA-construct contains sequences flanking the left and the right side of a naturally occurring deletion, e.g., deletion III, within the MVA genome. The foreign DNA sequence is inserted between the sequences flanking the naturally occurring deletion. For the expression of a DNA sequence or gene, it is necessary for regulatory sequences, which are required for the transcription of the gene, to be present on the DNA. Such regulatory sequences (called promoters) are known to those skilled in the art, and include for example those of the vaccinia 11 kDa gene as are described in EP-A- 198,328, and those of the 7.5 kDa gene (EP-A-1 10,385). The DNA-construct can be introduced into the MVA infected cells by transfection, for example by means of calcium phosphate precipitation (Graham et al. 1973 Virol 52:456-467; Wigler et al. 1979 Cell 16:777-785), by means of electroporation (Neumann et al. 1982 E B(9 J l:841-845), by microinjection (Graessmann et al. 1983 Meth Enzymol 101:482-492), by means of liposomes (Straubinger et al. 1983 Meth Enzymol 101:512-527), by means of spheroplasts (Schaffner 1980 PNAS USA 77:2163-2167) or by other methods known to those skilled in the art. The antigen to be encoded in respective priming and boosting compositions (however many boosting compositions are employed) can be, but need not be identical, but should share at least one CD8 T cell or antibody epitope. Designer sequences for shaping CD4 and CD8 T cell responses such as those representing consensus, conserved, or mosaic sequences may be employed. Designer sequences for eliciting cross-reactive antibody for consensus proteins, mosaic proteins or conserved regions of the proteins of a pathogen also may be employed. Furthermore, designer sequences targeting the elicitation of specific biological activities of antibody such as virus neutralization, antibody dependent cellular cytotoxicity (ADCC), and antibody dependent cell mediated inhibition of virus (ADCVI) may be used. Immune response modifiers such as cytokines (for example: granulocyte macrophage colony stimulating factor (GM-CSF), costimulatory molecules (for example B7 or CD40 ligand), or ligands for pattern recognition receptors that initiate immune responses (for example ligands for toll like receptors such as flagellin) may be incorporated into the same MVA as the vaccine antigens or into an MVA to be co-delivered with the MVA expressing the vaccine insert. Priming and boosting may be administered to the epidermis, intradermally, intramuscularly, or mucosally using devices developed for these deliveries such as microneedles, hypodermic needles, and

Biojecters . The MVA may be formulated with nanoparticles containing other immune response modifiers.

An HIV antigen of the invention to be encoded by a recombinant MVA virus includes polypeptides having immunogenic activity elicited by an amino acid sequence of an HIV Env, Gag, Pol (and optionally, Vif, Vpr, Tat, Rev, Vpu, or Nef) amino acid sequence as at least one CD8⁺ T cell or antibody epitope. This amino acid sequence substantially corresponds to at least one 10-900 amino acid fragment and/or consensus sequence of a known HIV Env or Pol; or at least one 10-450 amino acid fragment and/or consensus sequence of a known HIV Gag; or at least one 10-100 amino acid fragment and/or consensus sequence of a known HIV Vif, Vpr, Tat, Rev, Vpu, or Nef.

An HIV Env, Gag, or Pol can have overall identity at the amino acid or nucleic acid sequence of at least 80% to a known Env, Gag, or Pol protein amino acid sequence, such as 80- 99% identity, or any range or value therein, while eliciting an immunogenic response against at least one strain of an HIV. It may also be computer-generated mosaics of known HIV Gag, Pol, or Env proteins or computer- generated conserved sequences for known Gag, Pol, or Env proteins.

Percent identify can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (JMol Biol 1970 48:443), as revised by Smith and Waterman (Adv Appl Math 1981 2:482). Briefly, the GAP program defines identity as the number of aligned symbols {i.e., nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess (Nucl Acids Res 1986 14:6745), as described by Schwartz and Dayhoff (eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington, D.C. 1979, pp. 353-358); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

In a preferred embodiment, an Env of the present invention is a form of at least one HIV envelope protein. Preferably, the Env is composed of gpl20 and the complete membrane- spanning gp41 subunits of Env but may lack part or all of the cytoplasmic domain of gp41.

Known HIV sequences are readily available from commercial and institutional HIV sequence databases, such as GENBANK, or as published compilations, such as Myers et al. eds., Human Retroviruses and AIDS, A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, Vol. I and II, Theoretical Biology and Biophysics, Los Alamos, NM (2009 or 2010), or http://hiv-web.lanl.gov/.

Substitutions or insertions in a recombinant MVA to obtain expression of an HTV Env, Gag, or Pol or to obtain expression of an additional HIV Env, Gag, or Pol, can include substitutions or insertions in an existing gene of at least one amino acid residue (e.g., 1-25 amino acids). Alternatively, at least one amino acid (e.g., 1-25 amino acids) can be deleted from an HIV Env, Gag, or Pol sequence. Preferably, such substitutions, insertions or deletions are identified based on safety features, expression levels, immunogenicity and compatibility with high replication rates of MVA. Amino acid sequence variations in an HIV Env, Gag, or Pol of the present invention can be prepared e.g., by mutations in the DNA. Such HIV Env, Gag, or Pol include, for example, deletions, insertions or substitutions of nucleotides coding for different amino acid residues within the amino acid sequence. Obviously, mutations that will be made in nucleic acids encoding an HIV Env, Gag, or Pol must not place the sequence out of reading frame and preferably will not create instability of the vector.

HIV Env, Gag, or Pol-encoding nucleic acids of the present invention can also be prepared by amplification or site-directed mutagenesis of nucleotides in DNA or RNA encoding an HIV Env, Gag, or Pol and thereafter synthesizing the encoding DNA to produce DNA or RNA encoding an HIV Env, Gag, or Pol, based on the teaching and guidance presented herein. Sequences can also be mutated to eliminate 5TNT stop sites for vaccinia polymerases and codon optimized for enhanced expression

Thus, one of ordinary skill in the art, given the teachings and guidance presented herein, will know how to substitute amino acid residues in other positions of an HIV env, gag, or pol DNA or RNA to obtain alternative HIV Env, Gag, or Pol, including substitution, deletion or insertion variants as well as computer generated synthetic mosaic or conserved sequences.

Within the MVA vector, regulatory sequences for expression of the encoded antigen will include a natural, modified or synthetic poxvirus promoter. By "promoter" is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream {i.e. in the 3' direction on the sense strand of double-stranded DNA). "Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. Other regulatory sequences including terminator fragments, polyadenylation sequences, marker genes and other sequences may be included as appropriate, in accordance with the knowledge and practice of the ordinary person skilled in the art: see, for example, Moss, B. (2001). Poxviridae: the viruses and their replication. In Fields Virology, D.M. Knipe, and P.M. Howley, eds.

(Philadelphia, Lippincott Williams & Wilkins), pp. 2849-2883. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, 1998 Ausubel et al. eds., John Wiley & Sons.

Promoters for use in aspects and embodiments of the present invention must be compatible with poxvirus expression systems and include natural, modified and synthetic sequences.

The MVA composition may include an adjuvant, such as granulocyte macrophage- colony stimulating factor (GM-CSF) or encoding nucleic acid therefor.

The MVA used in each administration can be identical or different.

Administration of the boosting composition is generally about 1 to 10 months after administration of the priming composition, preferrably about 1 to 6 months, preferably about 1 ro 4 months, preferably about 1 to 3 months.

Preferably, administration of priming composition, boosting composition, or both priming and boosting compositions, is epidermal, intradermal, intramuscular or mucosal immunization.

Administration of MVA vaccines may be achieved by using a needle to inject a suspension of the virus. An alternative is the use of a needleless injection device such as Biojector™ needleless injector which can be used intramuscularly or intradermally.

Scarification and microneedles can be used for epidermal delivery. The MVA can be admistered as a virus suspension or a resuspended freeze-dried powder containing the vaccine, providing for manufacturing individually prepared doses that do not need cold storage. This would be a great advantage for a vaccine that is needed in rural areas of Africa.

Components to be administered in accordance with the present invention may be formulated in pharmaceutical compositions. These compositions may comprise a

pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. Physiological saline solution, sucrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For epidermal, cutaneous, subcutaneous, intramuscular or mucosal injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen- free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium

Chloride Injection, Ringer's Injection, Lactated Ringer's Injection or phosphate buffered saline. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as required.

Following production of MVA particles and optional formulation of such particles into compositions, the particles may be administered to an individual, particularly human or other primate.

In one regimen, MVA is administered at a dose of 10⁶ to 10⁹ infectious virus particles/injection.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

MVA recombinants expressing Clade A Env, Gag, and Pol

MVA 65A/G Construction and Characterization

One modified vaccinia virus Ankara (MVA) recombinant virus useful in the methods described herein is MVA 65A/G, which expresses clade A/G HIV strain 928 Env and Gag Pol. This MVA is described in US 2008/0193483. Features of this recombinant include:

1 ) The A/G env gene is inserted into del II of MVA genome and the A/G gag pol is inserted in del III.

2) Both env and gag pol of MVA 65 A/G are controlled by vaccinia PmH5 promoter.

3) The MVA virus used to make the recombinant MVA 65A/G is MVA 1974/NIH Clone 1.

The Gag/pol gene sequence of HIV A/G 928 contains three mutations to reduced the activity of reverse transcriptase activity (corresponding to that given for Clade B recombinant) and lacks integrase.

The Env gene sequence of HIV A/G 928 was truncated to remove 1 14 amino acids in the cytoplasmic tail of gp41 Silent mutations to eliminate two early poxvirus termination 5TNT signals were made. A variant of MVA 65 A/G (MVA 65 A/G Sma) was created by altering the region between the promoter for env and the env coding sequence. Briefly recloning of the envelope into a closer site to the promoter a intervening initiation codon was removed resulting in a virus which expressed larger quantities of env and was much more immunogenic.

MVA recombinants expressing Clade B Env, Gag, and Pol

MV A/HIV 62B Construction and Characterization

This example describes the construction of a modified vaccinia virus Ankara (MVA) recombinant virus, MV A/HIV 62B, expressing clade B HIV strains ADA Env and chimeric HXB2/BH10 Gag Pol. This MVA is described in US 2008/0193483.

This virus differs from an earlier MVA clade B recombinant, MV A/HIV 48, (which also expresses identical HTV strain ADA Env and HXB2/BH10 Gag Pol) in 4 ways:

1) MVA/HrV 62B uses a transient screening marker of green fluorescent protein (GFP) instead of GUS screening marker used in MV A/HIV 48.

2) The env gene is inserted into del Π of MVA genome and the gag pol is inserted in del III. In MV A/HIV 48, both env and gag pol are inserted into del ΠΙ.

3) Both env and gag pol of MV A/HIV 62B are controlled by PmH5, the same promoter controlling gag pol in MV A/HIV 48.

4) The MVA virus used to make the recombinant MV A/HIV 62B is MVA 1974/NIH Clone 1 instead of MVA 1983/NIH Clone 1 used to make MVA/HIV 48.

The clade B gag pol was truncated so that the integrase was removed and was cloned into the plasmid so that it was controlled by the mH5 promoter. This gene contained the complete HXB2 sequence of the gag. The pol gene has reverse transcriptase safety mutations in amino acid 185 within the active site of RT, in amino acid 266 which inhibits strand transfer activity, and at amino acid 478 which inhibits the Rnase H activity (numbering relative to the HXB2 HIV numbering standard). In addition, the integrase gene is deleted past EcoRI site.

The ADA envelope is a truncated version with silent 5TNT mutations. The envelope was truncated in the cytoplasmic tail of the gp41 gene, deleting 115 amino acids of the cytoplasmic tail. This truncation was shown by us to increase the amount of envelope protein on the surface of infected cells and enhance immunogenicity of the envelope protein in mice, and stability of the recombinant virus in tissue culture. MVA 56 Construction and Characterization

This example describes the construction of a modified vaccinia virus Ankara (MVA) recombinant virus, MVA/HIV clade B expressing HIV strain ADA env and chimeric

HXB2/BH10 gag pol. This MVA isUS 2008/0193483.

This virus differs from an earlier MVA recombinant, MVA/HIV 48, (which also expresses the HTV strain ADA env and the HXB2/BH10 gag pol) in 3 ways:

1. MVA/HIV 56 uses a transient screening marker of green fluorescent protein (GFP) instead of the GUS screening marker used in MVA/HIV 48.

2. The ADA env of MVA/HIV 56 is controlled by a new modified vaccinia virus promoter, Pm2H5, which allows more early expression of ADA env than the Psyn II promoter used to express the ADA env in MVA/HIV 48. (The gag pol is controlled by the vaccinia virus mH5promoter, the same promoter controlling the gag pol in MVA 48.)

3. The MVA virus used to make the recombinant MVA/HIV 56 is MVA

1974 NIH Clone 1 instead of MVA 1983/ IH Clone 1, used to make MVA HIV 48.

It differs from later constructs of original MVA 62B and modified MVA 62B in that the env and gag genes are controlled by double vaccinia virus promoters which are in tandem, like in MVA 48, and insert into only deletion III of the MVA genome. This gives this type of construct a construction advantage over the MVA 62B constructs since one has to go through only one set of plaque purifications as compared to the construction regime of the MVA 62B constructs.

All clade B constructs described have the same modified ADA env and modified HXB2/ BHIO gag pol.

MVA recombinant expressing Clade C Env, Gag, Pol

MVA/HIV 71 C Construction and Characterization

MVA/HIV 71C, expressing a clade C HTV IN3 Env and Gag Pol. This MVA is described in US 2003/0175292. The salient features of this recombinant virus are:

1. A transient screening marker of green fluorescent protein (GFP) was used in construction of MVA/HIV 71C, so that the GFP is eliminated in the final virus product.

2. The 71 C env gene is inserted into del II of MVA genome and the 71 C gag pol is inserted in del ΠΙ.

3. Both env and gag pol of MVA 71 C are controlled by vaccinia mH5 promoter. 4. The MVA virus used to make the recombinant MVA/HTV 71 C is MVA 1974/NIH Clone 1.

1. Lai L, Vodros D, Kozlowski PA, et al. GM-CSF DNA: An adjuvant for higher avidity IgG, rectal IgA, and increased protection against the acute phase of a SHIV-89.6P challenge by a DNA/MVA immunodeficiency virus vaccine. Virology 2007;369: 153-67

2. Kannanganant SN, P., Velu, V., Earl, P.L., Chennareddi, L., Lawson, B., Moss, B.,

Robinson, H.L., and Amara, RR. Preexisting Vaccinia Virus Immunity Influences the Quality of SrV-Specific T cells elicited by a DNA/MVA Vaccine and Enhances the Control of a Pathogenic SIV Challenge, submitted

3. Yokota A, Takeuchi H, Maeda N, et al. GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity. Int Immunol 2009;21 :361-77

4. Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C and Song SY. Retinoic acid imprints gut-homing specificity on T cells. Immunity 2004;21 :527-38

5. Mora JR, von Andrian UH. Retinoic acid: an educational "vitamin elixir" for gut-seeking T cells. Immunity 2004;21 :458-60

6. Zhao J, Lai L, Amara RR, et al. Preclinical studies of human immunodeficiency virus/ AIDS vaccines: inverse correlation between avidity of anti-Env antibodies and peak postchallenge viremia. J Virol 2009;83:4102-11

7. Aasa-Chapman MM, Holuigue S, Aubin K, et al. Detection of antibody-dependent complement-mediated inactivation of both autologous and heterologous virus in primary human immunodeficiency virus type 1 infection. J Virol 2005;79:2823-30

8. Sullivan BL, Knopoff EJ, Saifuddin M, et al. Susceptibility of HIV- 1 plasma virus to complement-mediated lysis. Evidence for a role in clearance of virus in vivo. J Immunol 1996;157: 1791-8

9. Ahmad A, Menezes J. Antibody-dependent cellular cytotoxicity in HIV infections. FASEB J 1996;10:258-66

10. Blumberg RS, Paradis T, Hartshorn KL, et al. Antibody-dependent cell-mediated cytotoxicity against cells infected with the human immunodeficiency virus. J Infect Dis 1987;156:878-84 11. Shepp DH, Chakrabarti S, Moss B and Quinnan GV, Jr. Antibody-dependent cellular cytotoxicity specific for the envelope antigens of human immunodeficiency virus. J Infect Dis 1988;157: 1260-4

12. Formal DN, Landucci G and Keenan B. Relationship between antibody-dependent cellular cytotoxicity, plasma HIV type 1 RNA, and CD4+ lymphocyte count. AIDS Res Hum

Retroviruses 2001;17:553-61

13. Schlesinger Y, Granoff DM. Avidity and bactericidal activity of antibody elicited by different Haemophilus influenzae type b conjugate vaccines. The Vaccine Study Group. JAMA 1992;267: 1489-94

14. Anttila M, Eskola J, Ahman H and Kayhty H. Avidity of IgG for Streptococcus

pneumoniae type 6B and 23F polysaccharides in infants primed with pneumococcal conjugates and boosted with polysaccharide or conjugate vaccines. J Infect Dis 1998;177: 1614-21

15. Parekh BS, Pau CP, Kennedy MS, Dobbs TL and McDougal JS. Assessment of antibody assays for identifying and distinguishing recent from long-term HIV type 1 infection. AIDS Res Hum Retroviruses 2001;17:137-46

16. Kim CN, Adams DR, Bashirian S, Butera S, Folks TM and Otten RA. Repetitive exposures with simian/human immunodeficiency viruses: strategy to study HIV pre-clinical interventions in non-human primates. J Med Primatol 2006;35:210-6

17. McDermott AB, Mitchen J, Piaskowski S, et al. Repeated low-dose mucosal simian immunodeficiency virus SIVmac239 challenge results in the same viral and immunological kinetics as high-dose challenge: a model for the evaluation of vaccine efficacy in nonhuman primates. J Virol 2004;78:3140-4

18. Keele BF, Li H, Learn GH, et al. Low-dose rectal inoculation of rhesus macaques by SIVsmE660 or SIVmac251 recapitulates human mucosal infection by HIV-1. J Exp Med 2009;206: 1 1 17-34

19. Yeh WW, Jaru-Ampornpan P, Nevidomskyte D, et al. Partial protection of Simian immunodeficiency virus (SlV)-infected rhesus monkeys against superinfection with a heterologous SW isolate. J Virol 2009;83:2686-96

20. Royce RA, Sena A, Cates WJ and Cohen MS. Current Concepts: Sexual Transmission of HIV. New England Journal of Medicine 1997;336: 1072-1078 21. Buge SL, Ma HL, Amara RR, et al. Gpl20-Alum Boosting of a Gag-Pol-Env DNA/MVA AIDS Vaccine: Poorer Control of a Pathogenic Viral Challenge. AIDS Res Hum Retroviruses 2003;19:891-900

22. Earl PL, Wyatt LS, Montefiori DC, et al. Comparison of vaccine strategies using recombinant env-gag-pol MVA with or without an oligomeric Env protein boost in the SHTV rhesus macaque model. Virology 2002;294:270-81

23. Robinson HL, Montefiori DC, Johnson RP, et al. Neutralizing Antibody-Independent Containment of Immunodeficiency Virus Challenges by DNA Priming and Recombinant Pox Virus Booster Immunization. Nature Medicine 1999;5:526-34

24. Smith JM, Amara RR, Campbell D, et al. DNA/MVA vaccine for HIV type 1 : effects of codon-optimization and the expression of aggregates or virus-like particles on the

immunogenicity of the DNA prime. AIDS Res Hum Retroviruses 2004;20:1335-47

25. Smith JM, Amara RR, McClure HM, et al. Multiprotein HIV-1 Clade B DNA/MVA Vaccine: Construction, Safety and Immunogenicity. AIDS Research and Human Retroviruses 2004;20:654-665

26. Wyatt LS, Earl PL, Liu JY, et al. Multiprotein HIV type 1 clade B DNA and MVA vaccines: construction, expression, and immunogenicity in rodents of the MVA component. AIDS Res Hum Retroviruses 2004;20:645-53

27. Wyatt LS, Earl PL, Vogt J, et al. Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 2008;26:486-93

28. Wyatt LS, Belyakov IM, Earl PL, Berzofsky JA and Moss B. Enhanced cell surface expression, immunogenicity and genetic stability resulting from a spontaneous truncation of HIV Env expressed by a recombinant MVA. Virology 2008;372:260-72

29. Bull M, Lee D, Stucky J, et al. Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 2007;322:57-69

30. McElrath MJ, De Rosa SC, Moodie Z, et al. HIV-1 vaccine-induced immunity in the test- of-concept Step Study: a case-cohort analysis. Lancet 2008;372:1894-905

31. Horton H, Thomas EP, Stucky JA, et al. Optimization and validation of an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-specific T cells induced by vaccination. J Immunol Methods 2007;323:39-54 32. Li F, Malhotra U, Gilbert PB, et al. Peptide selection for human immunodeficiency virus type 1 CTL-based vaccine evaluation. Vaccine 2006;24:6893-904

33. Perfetto SP, Chattopadhyay PK, Lamoreaux L, et al. Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 2006;313: 199-208

34. Barouch DH, O'Brien KL, Simmons NL, et al. Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat Med; 16:319-23

35. Amara RR, Villinger F, Altman JD, et al. Control of a Mucosal Challenge and Prevention of AIDS by a Multiprotein DNA/MVA Vaccine. Science 2001 ;292:69-74

36. Amara RR, Villinger F, Staprans S, et al. Different patterns of immune responses but similar control of a mucosal immunodeficiency virus challenge by MVA and DNA/MVA vaccines. Journal of Virology 2002;76:7625-7631

37. Ourmanov I, Brown CR, Moss B, et al. Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV. Journal of Virology 2000;74:2740-51

38. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA and Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. Journal of Immunology 1986;136:2348-57

39. Vermont CL, van Dijken HH, van Limpt CJ, de Groot R, van Alphen L and van Den Dobbelsteen GP. Antibody avidity and immunoglobulin G isotype distribution following immunization with a monovalent meningococcal B outer membrane vesicle vaccine. Infect Immun 2002;70:584-90

Claims

WHAT IS CLAIMED IS:

1. A method for eliciting an immune response in a subject, the method comprising administering a recombinant MVA encoding a HTV Env antigen, an HIV Gag antigen and an HIV Pol antigen wherein the HIV Env antigen comprises gpl20 and the membrane-spanning and ectodomain of gp41, wherein the recombinant MVA is administered at least three times.

2. The method of claim 1 wherein the subject is not administered a DNA vector expressing an HIV antigen.

3. The method of claim 1 wherein the administration of MVA occurs over a period of no more than 3, 6, 9, 12 or 24 months.

4. The method of claim 1 wherein the administration elicits IgA antibodies directed against HIV in rectal secretions of the subject.

5. The method of claim 1 wherein the administration elicits high avidity IgG to the native from of HIV.

6. The method of any of claim 1 wherein the administration elicits both CD4+ and CD8+ T cells.

7. The method of claim 1 wherein the administration elicits two or more of:

(i) IgA antibodies directed against HIV in rectal secretions of the subject; (ii) high avidity IgG to the native from of HIV; and (iii) CD4+ and CD8+ T cells.

8. The method of claim 1 wherein the administration elicits: (i) IgA antibodies directed against HTV in rectal secretions of the subject; (ii) high avidity IgG to the native from of HIV; and (iii) CD4+ and CD8+ T cells.

9. The method of claim 1 wherein the subject is a human.

10. The method of claim 1 the HTV Env antigen encoded by the recombinant MVA is at least 80% identical to any SEQ ID NOs: 7-11.

1 1. The method of claim 1 the HIV Gag antigen encoded by the recombinant MVA is at least 80% identical to any SEQ ID NOs: 12-15.

12. The method of claim 1 the HIV Pol antigen encoded by the recombinant MVA is at least 80% identical to any SEQ ID NOs: 16-18.

13. The method of claim 1 wherein the Env antigen comprises full-length gp41.

14. The method of claim 1 wherein the Pol antigen comprises fewer than the first 20 amino acids of p31 integrase.