WO2009051707A1 - Vaccine vectors - Google Patents

Abstract

The present invention relates, in general, to recombinant viral constructs expressing a protective antigen and to vaccine compositions comprising same. The invention further relates to a method of inducing an immune response, for example, to human immunodeficiency virus (HIV).

Description

VACCINE VECTORS

This application claims priority from U.S. Provisional Application No. 60/960,792, filed October 15, 2007, the entire content of which is incorporated herein by reference. This invention was made with government support under Grant No. U54

AI 057157 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to recombinant viral constructs expressing a protective antigen and to vaccine compositions comprising same. The invention further relates to a method of inducing an immune response, for example, to human immunodeficiency virus (HIV).

BACKGROUND

Designing vaccines for many infections, particularly an HIV vaccine, is a many- faceted challenge. The vaccine preferably elicits an immune response capable of either preventing infection or, minimally, controlling viral replication if infection occurs (Nabel, Vaccine 20:1945-1947 (2002)) or protecting from superinfection (Altfeld et al, Nature 420:434-439 (2002)). Potent vaccines are needed, with optimized vectors, immunization protocols, and adjuvants (Nabel, Vaccine 20: 1945-1947 (2002)), combined with antigens that can stimulate cross- reactive responses against the diverse spectrum of circulating viruses (Gaschen et al, Science 296:2354-2360 (2002), Korber et al, Br. Med. Bull. 58:19-42 (2001)). The present invention relates to viral constructs suitable for use as vaccine vectors for a number of infections, including, but not limited to, small pox, West Nile virus, HIV-I, TB, malaria, anthrax and plague. The vectors can be used, for example, in prime/boost regimens to induce protective and or therapeutic immune response to the encoded antigen(s).

SUMMARY OF THE INVENTION

The present invention relates to recombinant viral constructs expressing a protective antigen and to vaccine compositions comprising same. The invention further relates to a method of inducing an immune response, for example, to human immunodeficiency virus (HIV).

Objects and advantages of the present invention will be clear from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Verification of CPXV-B5 Expression. CPXV-B5 expression was verified in each vaccine vector (except M. smegmatis) via Western blotting for B5. Briefly, samples were prepared as appropriate for each vector (lysates of infected/tramsfected cells) and resolved by SDS-PAGE. No attempt was made to equalize protein loading between disparate samples (i.e. DNA and VRPs). Gel was transferred to nitrocellulose and blotted with heat inactivated polyclonal rabbit anti-sera (1 : 1000) in 5% nonfat dry milk/1 xTBST followed by goat anti rabbit IgG-AP 1 :3000 in 5% nonfat dry milk in IxTBST. Bands were visualized using Promega WesternBlue. Modified vaccinia Ankara (MVA, lane 5) was included as a positive control. The B5 expressed by MVA is the native non- truncated (42kD) form while the other vectors express the truncated 35kD form.

Figure 2. Primary responses to immunization with B5-expression vaccine vectors. Adult female C57BL/6 mice were immunized with a single intramuscular injection of B5 expressing vaccine vectors as indicated in Table 1. Six weeks after immunization all mice were bled and anti-B5 titers determined by binding ELISA. All sera were assayed individually. Endpoint titers were defined as the dilution reading 2x over background where background was the reading for pre-immune sera for each individual animal. Bars on graph are the geometric mean titer for each immunication group and error bars represent the upper and lower limits of the 95% confidence interval.

Figures 3A-3D. B5-immunized animals are protected from morbidity and mortality after sublethal intranasal challenge with wWR. Three months atfter Ad5 (Figs. 3A and 3C) or VSV (Figs. 3 B and 3D) boosting animals were challenged with a sublethal dose (1x10⁵ PFU) of vaccinia WR. Mice were monitored daily for mobidity and were euthanized if they lost more than 25% of their pre-challenge body weight. Figs. 3A and 3B show average daily weight for each immunization group, Figs. 3C and 3D show survival. Animals primed and boosted with MVA (orange triangles, Figs. 3 A and 3B) were completely protected from morbidity after challenge and served as a positive control for protection.

Figure 4. Intranasal-immunized animals are protected from morbidity and mortality after sublethal intranasal challenge with wWR. Three months after the single boost animals were challenged with a sublethal dose (1x10⁵ PFU) of vaccinia WR. Mice were monitored daily for morbidity and were euthanized if they lost more than 25% of their pre-challenge body weight. Legend indicates prime/boost vector for each group. Graph shows average daily weight by group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention results, in part, from studies demonstrating that the induction of an immune response can be enhanced when vaccination is performed utilizing certain recombinant viral constructs. In a preferred embodiment, the invention relates to recombinant viral constructs comprising a nucleic acid molecule encoding one or more HIV antigens.

In one aspect, the present invention relates to a recombinant viral construct comprising a vesicular stomatitis virus (VSV) vector, or functional derivative thereof, which comprises a nucleic acid molecule encoding one or more HIV antigens, or derivatives thereof, wherein the recombinant viral construct is effective in inducing, enhancing or otherwise stimulating an immune response to the HIV antigen.

In a further aspect, the present invention relates to a recombinant viral construct comprising a Venezuelan equine encephalitis virus replicon particle (VEE-VRP) vector, or functional derivative thereof, which comprises a nucleic acid molecule encoding one or more HIV antigens, or derivatives thereof, wherein the recombinant viral construct is effective in inducing, enhancing or otherwise stimulating an immune response to the HIV antigen.

Examples of antigens suitable for use in the present invention include, but are not limited to, one or more of the molecules encoded by the HIV viral genes (gag, pol, nef, env, tat, rev, etc), or antigenic portions thereof. Preferred HIV-I antigens include consensus or mosaic sequences described, for, example, in US Application Nos. 10/572,638 (see also PCT/US2004/030397), 11/896,934 and PCT/US2006/032907, or signature sequences described, for example, in US Provisional Applications 60/907,259 and 60/924,398. While the invention is described in detail with reference to HIV-I, the invention includes viral constructs comprising antigens other than HIV antigens. For example, the invention includes vaccine vectors suitable for use in inducing immune responses to small pox, West Nile virus, HIV-I, TB, malaria, anthrax and plague and methods based on same.

Compositions comprising the viral constructs of the invention and a suitable carrier can be administered in any convenient manner, such as by the oral, intravenous, intranasal, intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g., using slow release molecules). Compositions suitable for injectable use include sterile aqueous solutions. Compositions suitable for use in the invention can be in dosage unit form.

Still another aspect of the present invention relates to a method of inducing, enhancing or otherwise stimulating an immune response, in a mammal (human or non-human), to, for example, HIV. The method comprises administering to the mammal an amount of the recombinant viral constructs for a time and under conditions sufficient to induce, enhance or otherwise stimulate the desired immune response (e.g., to one or more HIV antigens).

The method of the present invention is useful in the treatment and/or prophylaxis of HIV infection and AIDS. For example, the vaccine of the present invention can be administered into subjects known to be infected with HIV in order induce an immune response against HIV, thereby preventing the onset of AIDS. Alternatively, the method of the present invention may be used to reduce serum viral load, to alleviate AIDS symptoms or to induce immunity in mammals thought to be at risk of HIV infection.

Optimum dosage strategies for inducing the desired response can be readily determined by one skilled in the art and can vary with the constructs, the patient and the effect sought. Preferred prime/boost regimens include an rVSV IM prime with a VEE(VRP) IM boost or an intra nasal prime and boost with the same vectors. Recombinant Ad 5 can be used as a prime for either rVEE or rVSV as well. For HIV-I to overcome diversity, the preferred inserts in rVSV and rVEE are mosiac or consensus Env genes (e.g., 3-4 mosiacs, or one consensus) and, for eample, 2-4 mosaic gag, pol, nef and tat genes.

Certain aspects of the invention can be described in greater detail in the non-limiting Example that follows.

EXAMPLE 1

A comparison has been made of the efficacy of various vaccine vectors. Vaccine vectors all contain the same insert (poxvirus protein CPXV B5) and are being evaluated for immunogenicity and the ability to protect mice from poxvirus challenge. The CPXV B5 insert is a truncated (secreted) form of CPXV-B5 from which the transmembrane and cytoplasmic domains have been deleted (B5Oct279).

Materials for expression of poxvirus protein B5R: 1. Venezuelan equine encephalitis virus replicon particles (VEE-VRP)

(Thornburg et al, Virology 362:441 (2007))

2. rAd5 (Santra et al, J. Virol. 79(10):6516-22 (2005))

3. Attenuated rVSV (Lawson et al, Proc. Natl. Acad. Sci. 92:4477 (1995)

4. M. smegmatis (Yu et al, CHn. Vaccine Immunol. 13(1 l):1204-l 1 (2006) Epub Aug. 30, 2006)

5. As purified protein

6. As DNA in vector pCMVR

Immunization of mice with Modified vaccinia Ankara (MVA) is known to protect against vaccinia challenge in mice. This protection is dependent upon responses to multiple poxvirus proteins, including, but not limited to, B5. Mice immunized with MVA (which expresses B5 in the native form) will be included as a positive control for immune responses and protection from challenge.

Vector Construction and Characterization. The B5 containing vectors listed above have been constructed. Expression of the B5Oct279 insert has been verified for all constructs. A Western blot showing expression of B5 by all vectors is shown in Fig. 1. The M. smegmatis vector produced B5 at low levels and expression could not be confirmed by western blotting. Expression was confirmed instead by sequencing of the vector (data not shown).

Results

Primary intramuscular immunization with vaccine vectors expressing CPXV B5 induces high titer anti-B5 serum antibody. To determine whether vaccination with B 5 expressing vaccine vectors could induce primary immune responses in mice, adult female C57BL/6 mice were immunized with each vector and serum antibody titers to B5 were measured via binding ELISA. Dosing and immunization schedule are summarized in Table 1. In order to standardize the immunization protocol, all immunizations were administered intramuscularly in a single 50μl injection for which the diluent was sterile saline. Thus, for all vectors, the only variable was dose and vector. Dosing for each vector was based, in part, on previous optimization experiments. For rVSV and the VRP, the dose was limited by the amount of virus that could be delivered in the 50μl inoculation volume. The experiment as outlined in Table 1 has been done twice with consistent results. Data in subsequent figures and tables includes combined results from the two duplicate experiments.

*A11 immunizations given IM in 50μl total volume (quadriceps). Protein administered with adjuvant formulated for IM injection (Emulsigen-oligo CpG) * Animals were boosted six weeks after primary immunization.

Primary humoral responses to B5 were assayed at four (Table 2) and six (Table 2, Fig. 2) weeks post immunization. All vectors induced robust anti-B5 serum antibody titers except for M. smegmatis. Anti-B5 antibodies were not detected in any of the 48 animals immunized with M. smegmatis. This was likely due to the low level of expression of the B5 protein by this vector. rAd5 induced the highest antibody titers, followed by recombinant protein. rVSV, VRP, MVA, and DNA all induced similar and lower levels of anti-B5 antibody. Most animals developed measurable anti-B5 titers by four weeks post immunization, with titers increasing slightly by six weeks. There were no significant differences in kinetics between immunization groups.

Table 2: Primary responses to intramuscular immunization with B5 expressing vaccine vectors

- - -Heterologous boosting increases. serum antibody -titers to B 5. Six weeks after the single primary immunization, animals were boosted with either rAd5-B5 or rVSV-B5 as indicated in Table 1. Serum was collected at two and four weeks after boosting, then once monthly every month for three months after the boost. Antibody titers rose significantly after boosting and remained elevated for the three months after the boost. Table 3 summarizes the average peak titers reached for each group in the three month period.

Table 3: Recall responses after heterologous boosting

^♦Numbers in table are the geometric mean titer for each group. Range indicates values falling within the 95% confidence interval. Post boost responses fell into three broad categories indicated by the shading in Table 3. An ideal boost should raise titers above those generated by priming, as well as above those generated by the boosting vector alone. The top three groups (darker shading, Table 3) as well as the VRP prime/VSV boost group (lighter shading) satisfied these criteria and were the "best" prime-boost combinations. In contrast, anti-B5 titers of animals pnmed with VSV, DNA, VRP, or MVA and boosted with rAd5 did not πse significantly above titers generated by rAd5 pπming alone (rAd5 priming titer=39,037, Table 2). This indicates that a single intramuscular immunization with rAd5 is enough to confer close to maximal titers and may also be sufficient to provide protection. rVSV boosting efficiently increased anti-B5 titers regardless of priming vector, with VRP-VSV being a particularly synergistic combination. M, smegmatis primed animals had generally weak post-boosting responses, and, in the case of M, smeg/rAd5, the M. smegmatis prime may actually have inhibited the recall response.

Mice immunized with B5-expressing vectors are protected from sublethal challenge with vaccinia virus WR. Three months after boosting all mice were challenged intranasally with a sublethal dose ofwWR. Most animals exhibited transient weight loss which resolved by one to two weeks post challenge. Average daily weight and survival by group is shown in Figs. 3A and C (rAd5 boosted mice) and Figs. 3B and D (VSV boosted mice). Animals having higher anti-B5 titers were better protected from morbidity/mortality after challenge on both a group and an individual level. It is not possible at present to exclude the possibility that anti-B5 T cell responses contribute to the observed protection, however, these data suggest that anti-B5 Ab may be sufficient to protect from (sub)lethal poxvirus challenge. Passive transfer experiments are being undertaken to confirm the role of anti-B5 antibody in the absence of anti-B5 T cell responses. Passive transfer experiments will make it possible to elucidate qualitative differences in the antibody responses generated by the different prime-boost combinations. An experiment to test whether a single immunization with either of the boosting vectors alone (rAd5 or rVSV) is sufficient to confer protection is currently underway. Based on preliminary experiments and the anti-B5 titers generated by rAd5 and rVSV in this experiment, it is predicted that rAd5-B5, but not rVSV-B5, will confer protection.

Primary intranasal immunization with vaccine vectors expressing CPXV B 5 induces higher anti-B5 titers than does intramuscular immunization. Many of the infectious agents of concern for deliberate release and/or bioterrorism are spread by aerosol or droplet (variola, influenza, anthrax). Accordingly, it is important to determine which vaccine vectors are capable of generating protective antibody at the mucosal/respiratory surfaces which are the site of initial infection with these agents. It was hypothesized that intranasal delivery of the most immunogenic vectors identified in the intramuscular trial might enhance immunogenicity of these vectors and generate protective antibody (IgG or IgA) in the respiratory tract. rAd5, rVSV, and VRP vectors were tested in an intranasal trial (ongoing) as outlined in Table 4. Total dose volume for all immunizations was 30μl.

Table 4. Intranasal vaccine vector comparison

Primary serum antibody responses to B5 were assayed via binding ELISA at six weeks post immunization (Table 5). In all cases antibody titers were significantly higher after intranasal immunization than after intramuscular immunization (compared in Table 5). As in the intramuscular trial, rAd5 induced the highest serum Ab titers to B5 after primary immunization. Titers induced by rVSV and VRP vectors were nearly 10x higher than those induced by intramuscular immunization. Consistent with the results of the intramuscular trial, rVSV and VRP were the most synergistic prime-boost combination, inducing titers 100Ox higher than those induced by priming with either vector alone. Taken together, these data indicate that intranasal immunization may be a preferable route for vaccination against some agents and may allow induction of greater antibody responses with a smaller amount of antigen (dose sparing). Table 5. Comparison of serum titers: Intramuscular versus intranasal administration

Heterologous boosting increases serum antibody titers to B5. Six weeks after the single primary immunization animals were boosted intranasally with either rAd5-B5, rVSV-B5, or VRP-B5 as indicated in Table 4. Serum was collected at two and four weeks after boosting, then once monthly every month for three months after the boost. Serum antibody titers rose significantly after boosting and remained elevated for the three months after the boost. Table 6 summarizes the average peak titers reached for each group in the three month period.

Table 6: Anti-B5 serum antibody titers after heterologous boosting

Mice immunized with B 5 -expressing vectors are protected from sublethal challenge with vaccinia virus WR. Three months after boosting all mice were challenged intranasally with a sublethal dose of wWR (Figure 4). This experiment is ongoing but, in contrast to the challenge after intramuscular immunization, in which most animals exhibited considerable morbidity, some intranasally immunized animals were completely protected from weight loss after challenge (highlighted groups in Table 6).

In conclusion, a single intramuscular immunization with rAd5-B5 induces near maximal anti-B5 serum antibody titers and may be sufficient to confer protection from challenge, further, heterologous boosting of rAd5-primed animals generally does not significantly raise antibody titers above those conferred by priming alone and may be unnecessary for vaccine efficacy. M. smegmatis-B5 was non-immunogenic in the present study and did not synergistically prime for an rAd5 or rVSV boost. rVSV was an effective boosting vector for all priming immunogens except M. smegmatis. rVSV and VRP were a highly synergistic prime-boost combination when delivered by the intramuscular or intranasal route. Finally, intranasal delivery improved serum antibody titers to all vectors tested relative those generated by intramuscular immunization. Intranasal delivery of vaccines may allow induction of higher titer antibodies with a smaller amount of antigen (dose sparing) relative intramuscular immunization. (The data presented above has not yet been subjected to detailed statistical analysis.)

EXAMPLE 2

Experimental Details:

Generation of vaccine vectors. Recombinant vesicular stomatitis virus (rVSV), recombinant Venezuelan equine encephalitis virus replicon particles (VRP), and recombinant adenovirus expressing the vaccine antigen CON-S gpl40CF will be generated as follows:

rVSV CON-Sgpl40 CF. To obtain a plasmid that can be used to recover an rVSV expressing CON-S gpl40 from the fifth position in the VSV genome, the DNA fragment encoding CON-S gpl40CF was PCR amplified and cloned into the pVS V XN2 expression vector (provided by Dr. John Rose, Yale University) (Lawson et al, Proc. Natl. Acad. Sci. 92:4477 (1995)). The forward primer introduced an Xhol site upstream of the CON-S gpl40 coding sequence while the reverse inserted a Nhel site downstream. The PCR product was digested with Xhol and Nhel, purified, and ligated into the pVSV XN2 vector, which had been digested with the same enzymes to generate pVSV XN2 CON-S gpl40. Plasmids were recovered after the transformation of Escherichia coli and were purified using a Maxi kit (QIAGEN). The insert sequence was verified (Duke Sequencing Facility). Recombinant virus was recovered from these plasmids as described previously. Briefly, BHK-21 cells were grown to 50% confluence and infected at a multiplicity of infection (MOI) of 10 with vTF7-3, a vaccinia virus expressing T7 RNA polymerase. One hour after infection, cells were transfected with 10 μg of pVSV XN2 CON-S gpl40 along with 3 μg of pBS-N, 5 μg of pBS-P, 1 μg of pBS-L, and 4 μg of pBS-G. After 48 h, cell supernatants were passaged onto BHK-21 cells through a 0.2-μm filter, and medium containing virus was collected about 24 h after the cytopathic effect was seen. Viruses grown from individual plaques were used to prepare stocks that were grown on BHK-21 cells and stored at 80⁰C.

VRP-CON-S gp 140 CF. VRP production has been described previously ^• (Davis et al, J. Virol. 74(1) 371-8 (2000), Erratum in J. Virol. 74(7):3430 (2000)) Briefly, the coding region of the CON-S gene will be PCR amplified from plasmid DNA and inserted into VEE replicon plasmid pVR21 (Pushko, Virol. 239(2):389-401 (1997)). Next, pVR21 encoding the VEE nonstructural proteins, an encapsidation signal, and the transgene downstream of a 26S promoter, and two helper plasmids encoding VEE capsid and VEE E1/E2 glycoproteins will be linearized and transcribed using the mMessage in vitro transcription kit (Ambion). The attenuated V3014 glycoproteins were used to package all VRP (Bernard et al Virology 276:93 (2000)). Transcripts will be electroporated into BHK cells. VRP will be recovered from culture superaatants and purified by ultra- centrifugation through a 20% sucrose cushion. Concentrated VRP will be resuspended in PBS and stored at -80°.

r AdS-CON-S gp 140. The CON-S gpHOCF gene was subcloned into the Ad5 adaptor plasmid pAdApt. El/E3-deleted rAd5-CON-S gpl40 CF vectors were produced by transfection of complementing 293 cells with pAdApt-B5R and the structural cosmid pWE.Ad.AflllrlTR with lipofectamine in T25 flasks. Cells were passaged into T75 flasks after 48 hrs and maintained until virus cytopathic effect was observed. The vectors were plaque-purified, analyzed for transgene expression, amplified in 24 triple-layer Tl 75 flasks, purified by double CsCl gradient ultracentrifugation, and dialyzed into PBS containing 5% sucrose. Purified rAd vectors were stored at -80⁰C. Virus particle (vp) titers were determined by spectrophotometry. Specific infectivity was assessed by plaque forming unit (pfu) assays. Immunization and comparative immunogenicity studies The goal of this experiment is to determine the optimal prime-boost combination for induction of anti-HIV Env binding and/or neutralizing antibodies. Guinea pigs will be immunized with the CON-S gpl40CF expressing vectors according to the plan shown in Table 7below. All animals will be pre-bled prior to beginning the study, with additional blood collections at 2 weeks post primary immunization, 4 weeks post primary immunization, 6 weeks post primary immunization, and then at 2, 4, 8, and 12 weeks after boosting. All animals will be immunized intramuscularly, and will receive a single injection in the rear quadriceps of 200μl total volume. All animals will receive a single primary immunization, followed by a single booster immunization at six weeks post boost.

Table 7 Vaccine vector comparison experiment 1

Measurement of humoral immune responses. Antibodies to HIV Env will be quantified in the sera of immunized animals via binding ELISA and by standard neutralizing assay. The use of these two assays will make it possible to determine the absolute titer of antibodies generated by immunization (binding ELISA) as well as their breadth and potential to neutralize infectious virus in vivo (neutralizing assay). All documents and other information sources cited above are hereby incorporated in their entirety by reference.

Claims

WHAT IS CLAIMED IS:

1. A recombinant viral construct comprising a vesicular stomatitis virus (VSV) vector, or functional derivative thereof, which comprises a nucleic acid molecule encoding at least one antigen.

2. The construct according to claim 1 wherein said antigen is an HIV, small pox, West Nile virus, TB, malaria, anthrax or plague antigen.

3. The construct according to claim 2 wherein said antigen is an HIV antigen.

4. The construct according to claim 3 wherein said nucleic acid molecule encodes gag, pol, nef, env, tat or rev, or antigenic portion thereof.

5. A recombinant viral construct comprising a Venezuelan equine encephalitis virus replicon particle (VEE-VRP) vector, or functional derivative thereof, which comprises a nucleic acid molecule encoding at least one antigen.

6. The construct according to claim 5 wherein said antigen is an HIV, small pox, West Nile virus, TB, malaria, anthrax or plague antigen.

7. The construct according to claim 6 wherein said antigen is an HIV antigen.

8. The construct according to claim 7 wherein said nucleic acid molecule encodes gag, pol, nef, env, tat or rev, or antigenic portion thereof.

9. A composition comprising the construct of claim 1 or 5 and a carrier.

10. A method of inducing or enhancing an immune response in a mammal to and antigen comprising administering to said mammal an amount of said construct according to claim 1 or 5 in an amount sufficient to induce or enhance said immune response

1 1. The method according to claim 10 wherein said antigen is an HIV, small pox, West Nile virus, TB, malaria, anthrax or plague antigen.

12. The method according to claim 11 wherein said antigen is an HIV antigen.