Single-Injection Vaccine Protects Nonhuman Primates against Infection with Marburg Virus and Three Species of Ebola Virus

The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Cote d''Ivoire ebolavirus (CIEBOV), and Marburgvirus (MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVΔG/SEBOVGP, VSVΔG/ZEBOVGP, and VSVΔG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVΔG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic filovirus species.Marburgvirus (MARV) and Ebolavirus (EBOV), the causative agents of Marburg and Ebola hemorrhagic fever (HF), respectively, represent the two genera that comprise the family Filoviridae (8, 24). The MARV genus contains a single species, Lake Victoria marburgvirus. The EBOV genus is divided into four distinct species: (i) Sudan ebolavirus (SEBOV), (ii) Zaire ebolavirus (ZEBOV), (iii) Cote d''Ivoire ebolavirus (CIEBOV), and (iv) Reston ebolavirus (REBOV). A putative fifth species of EBOV was associated with an outbreak in Uganda late in 2007 (33). MARV, ZEBOV, and SEBOV are important human pathogens, with case fatality rates frequently ranging between 70% and 90% for ZEBOV, around 50% for SEBOV, and up to 90% for MARV outbreaks depending on the strain of MARV (reviewed in reference 24). CIEBOV caused deaths in chimpanzees and a severe nonlethal human infection in a single case in the Republic of Cote d''Ivoire in 1994 (21). REBOV is highly lethal for macaques but is not thought to cause disease in humans, although the pathogenic potential of REBOV in humans remains unknown (24). An outbreak of REBOV in pigs was recently reported in the Philippines; however, it is unclear whether the disease observed in the pigs was caused by REBOV or other agents detected in the animals, including porcine reproductive and respiratory syndrome virus (5, 22).While there are no FDA-approved vaccines or postexposure treatment modalities available for preventing or managing EBOV or MARV infections, there are at least five different vaccine systems that have shown promise in completely protecting nonhuman primates against EBOV, and four of these systems have also been shown to protect macaques against MARV HF (3, 6, 12, 18, 20, 28-31, 35). Several of these vaccine platforms require multiple injections to confer protective efficacy (3, 18, 30, 31, 35). However, for agents such as EBOV and MARV, which are indigenous to Africa and are also potential agents of bioterrorism, a single-injection vaccine is preferable. In the case of preventing natural infections, multiple-dose vaccines are both too costly and not practicable (logistics and compliance) in developing countries. In the case of a deliberate release of these agents, there would be little time for deployment of a vaccine that requires multiple injections. Thus, for most practical applications, a vaccine against the filoviruses necessitates a single immunization.Of the prospective filovirus vaccines, only two systems, one based on a replication-defective adenovirus serotype 5 and the other based on the recombinant vesicular stomatitis virus (VSV), were shown to provide complete protection to nonhuman primates when administered as a single-injection vaccine (6, 12, 20, 28, 29). Most intriguingly, the VSV-based vaccine is the only vaccine which has shown utility when administered as a postexposure treatment against filovirus infections (7, 9, 15). Here, we evaluated the utility of combining our VSV-based EBOV and MARV vectors into a single-injection vaccine and determined the ability of this blended vaccine to protect nonhuman primates against three species of EBOV and MARV. Furthermore, we assessed the reusability of the VSV vectors in our macaque models of filovirus HF.     

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus

Background

Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the ‘bush-meat’ trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes.

Methodology/Principal Findings

We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a ‘proof-of-concept’ for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8⁺ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP_CTL). MCMV/ZEBOV-NP_CTL induced high levels of long-lasting (>8 months) CD8⁺ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection.

Conclusions/Significance

This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for ‘disseminating’ CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.     

Vesicular Stomatitis Virus-Based Vaccines Protect Nonhuman Primates against Bundibugyo ebolavirus

Ebola virus (EBOV) causes severe and often fatal hemorrhagic fever in humans and nonhuman primates (NHPs). Currently, there are no licensed vaccines or therapeutics for human use. Recombinant vesicular stomatitis virus (rVSV)-based vaccine vectors, which encode an EBOV glycoprotein in place of the VSV glycoprotein, have shown 100% efficacy against homologous Sudan ebolavirus (SEBOV) or Zaire ebolavirus (ZEBOV) challenge in NHPs. In addition, a single injection of a blend of three rVSV vectors completely protected NHPs against challenge with SEBOV, ZEBOV, the former Côte d''Ivoire ebolavirus, and Marburg virus. However, recent studies suggest that complete protection against the newly discovered Bundibugyo ebolavirus (BEBOV) using several different heterologous filovirus vaccines is more difficult and presents a new challenge. As BEBOV caused nearly 50% mortality in a recent outbreak any filovirus vaccine advanced for human use must be able to protect against this new species. Here, we evaluated several different strategies against BEBOV using rVSV-based vaccines. Groups of cynomolgus macaques were vaccinated with a single injection of a homologous BEBOV vaccine, a single injection of a blended heterologous vaccine (SEBOV/ZEBOV), or a prime-boost using heterologous SEBOV and ZEBOV vectors. Animals were challenged with BEBOV 29–36 days after initial vaccination. Macaques vaccinated with the homologous BEBOV vaccine or the prime-boost showed no overt signs of illness and survived challenge. In contrast, animals vaccinated with the heterologous blended vaccine and unvaccinated control animals developed severe clinical symptoms consistent with BEBOV infection with 2 of 3 animals in each group succumbing. These data show that complete protection against BEBOV will likely require incorporation of BEBOV glycoprotein into the vaccine or employment of a prime-boost regimen. Fortunately, our results demonstrate that heterologous rVSV-based filovirus vaccine vectors employed in the prime-boost approach can provide protection against BEBOV using an abbreviated regimen, which may have utility in outbreak settings.     

Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates

Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4⁺ and CD8⁺ T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4⁺ T-cell response (NYVAC). Remarkably, vector-induced differences in CD4⁺/CD8⁺ T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4⁺ T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4⁺ T-cell responses showed efficacies similar to those with stronger CD8⁺ T-cell responses.     

Immunization with Single-Cycle SIV Significantly Reduces Viral Loads After an Intravenous Challenge with SIVmac239

Strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection were evaluated for the ability to elicit protective immunity against wild-type SIV_mac239 infection of rhesus macaques by two different vaccine regimens. Six animals were inoculated at 8-week intervals with 6 identical doses consisting of a mixture of three different envelope variants of single-cycle SIV (scSIV). Six additional animals were primed with a mixture of cytoplasmic domain-truncated envelope variants of scSIV and boosted with two doses of vesicular stomatitis virus glycoprotein (VSV G) trans-complemented scSIV. While both regimens elicited detectable virus-specific T cell responses, SIV-specific T cell frequencies were more than 10-fold higher after boosting with VSV G trans-complemented scSIV (VSV G scSIV). Broad T cell recognition of multiple viral antigens and Gag-specific CD4⁺ T cell responses were also observed after boosting with VSV G scSIV. With the exception of a single animal in the repeated immunization group, all of the animals became infected following an intravenous challenge with SIV_mac239. However, significantly lower viral loads and higher memory CD4⁺ T cell counts were observed in both immunized groups relative to an unvaccinated control group. Indeed, both scSIV immunization regimens resulted in containment of SIV_mac239 replication after challenge that was as good as, if not better than, what has been achieved by other non-persisting vaccine vectors that have been evaluated in this challenge model. Nevertheless, the extent of protection afforded by scSIV was not as good as typically conferred by persistent infection with live, attenuated SIV. These observations have potentially important implications to the design of an effective AIDS vaccine, since they suggest that ongoing stimulation of virus-specific immune responses may be essential to achieving the degree of protection afforded by live, attenuated SIV.     

Oral Immunization of Macaques with Attenuated Vaccine Virus Induces Protection against Vaginally Transmitted AIDS

The chimeric simian-human immunodeficiency virus SHIV_KU-1, bearing the envelope of human immunodeficiency virus type 1 (HIV-1), causes fulminant infection with subtotal loss of CD4⁺ T cells followed by development of AIDS in intravaginally inoculated macaques and thus provides a highly relevant model of sexually transmitted disease caused by HIV-1 in human beings. Previous studies using this SHIV model had shown that the vpu and nef genes were important in pathogenesis of the infection, and so we deleted portions of these genes to create two vaccines, ΔvpuΔnefSHIV-4 (vaccine 1) and ΔvpuSHIV_PPc (vaccine 2). Six adult macaques were immunized subcutaneously with vaccine 1, and six were immunized orally with vaccine 2. Both viruses caused infection in all inoculated animals, but whereas vaccine 1 virus caused only a nonproductive type of infection, vaccine 2 virus replicated productively but transiently for a 6- to 10-week period. Both groups were challenged 6 to 7 months later with pathogenic SHIV_KU-1 by the intravaginal route. All four unvaccinated controls developed low CD4⁺ T-cell counts (<200/μl) and AIDS. The 12 vaccinated animals all became infected with SHIV_KU-1, and two in group 1 developed a persistent productive infection followed by development of AIDS in one. The other 10 have maintained almost complete control over virus replication even though spliced viral RNA was detected in lymph nodes. This suppression of virus replication correlated with robust antiviral cell-mediated immune responses. This is the first demonstration of protection against virulent SHIV administered by the intravaginal route. This study supports the concept that sexually transmitted HIV disease can be prevented by parenteral or oral immunization.     

A Chimeric Vesiculo/Alphavirus Is an Effective Alphavirus Vaccine

While a large number of mosquito-transmitted alphaviruses are known to cause serious human diseases, there are no licensed vaccines that protect against alphavirus infections. The alphavirus chikungunya virus (CHIKV) has caused multiple recent outbreaks of chikungunya fever. This virus has the potential to cause a worldwide epidemic and has generated strong interest in development of a prophylactic CHIKV vaccine. We report here on the development of a potent experimental vaccine for CHIKV based on a chimeric vesicular stomatitis virus (VSV) expressing the entire CHIKV envelope polyprotein (E3-E2-6K-E1) in place of the VSV glycoprotein (G). These VSVΔG-CHIKV chimeras incorporated functional CHIKV glycoproteins into the viral envelope in place of VSV G. The chimeric viruses were attenuated for growth in tissue culture but could be propagated to high titers without VSV G complementation. They also generated robust neutralizing antibody and cellular immune responses to CHIKV in mice after a single dose and protected mice against CHIKV infection. VSVΔG-alphavirus chimeras could have general applicability as alphavirus vaccines.     

Recombinant vesicular stomatitis virus vaccine vectors expressing filovirus glycoproteins lack neurovirulence in nonhuman primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use.     

Inhibition of Simian Immunodeficiency Virus (SIV) Replication by CD8+ T Lymphocytes from Macaques Immunized with Live Attenuated SIV

Characterization of immune responses induced by live attenuated simian immunodeficiency virus (SIV) strains may yield clues to the nature of protective immunity induced by this vaccine approach. We investigated the ability of CD8⁺ T lymphocytes from rhesus macaques immunized with the live, attenuated SIV strain SIVmac239Δnef or SIVmac239Δ3 to inhibit SIV replication. CD8⁺ T lymphocytes from immunized animals were able to potently suppress SIV replication in autologous SIV-infected CD4⁺ T cells. Suppression of SIV replication by unstimulated CD8⁺ T cells required direct contact and was major histocompatibility complex (MHC) restricted. However, CD3-stimulated CD8⁺ T cells produced soluble factors that inhibited SIV replication in an MHC-unrestricted fashion as much as 30-fold. Supernatants from stimulated CD8⁺ T cells were also able to inhibit replication of both CCR5- and CXCR4-dependent human immunodeficiency virus type 1 (HIV-1) strains. Stimulation of CD8⁺ cells with cognate cytotoxic T-lymphocyte epitopes also induced secretion of soluble factors able to inhibit SIV replication. Production of RANTES, macrophage inhibitory protein 1α (MIP-1α), or MIP-1β from stimulated CD8⁺ T cells of vaccinated animals was almost 10-fold higher than that from stimulated CD8⁺ T cells of control animals. However, addition of antibodies that neutralize these β-chemokines, either alone or in combination, only partly blocked inhibition of SIV and HIV replication by soluble factors produced by stimulated CD8⁺ T cells. Our results indicate that inhibition of SIV replication by CD8⁺ T cells from animals immunized with live attenuated SIV strains involves both MHC-restricted and -unrestricted mechanisms and that MHC-unrestricted inhibition of SIV replication is due principally to soluble factors other than RANTES, MIP-1α, and MIP-1β.     

Mucosal Immunization of Cynomolgus Macaques with the VSVΔG/ZEBOVGP Vaccine Stimulates Strong Ebola GP-Specific Immune Responses

Background

Zaire ebolavirus (ZEBOV) produces a lethal viral hemorrhagic fever in humans and non-human primates.

Methodology/Principal Findings

We demonstrate that the VSVΔG/ZEBOVGP vaccine given 28 days pre-challenge either intranasally (IN), orally (OR), or intramuscularly (IM) protects non-human primates against a lethal systemic challenge of ZEBOV, and induces cellular and humoral immune responses. We demonstrated that ZEBOVGP-specific T-cell and humoral responses induced in the IN and OR groups, following an immunization and challenge, produced the most IFN-γ and IL-2 secreting cells, and long term memory responses.

Conclusions/Significance

We have shown conclusively that mucosal immunization can protect from systemic ZEBOV challenge and that mucosal delivery, particularly IN immunization, seems to be more potent than IM injection in the immune parameters we have tested. Mucosal immunization would be a huge benefit in any emergency mass vaccination campaign during a natural outbreak, or following intentional release, or for mucosal immunization of great apes in the wild.     

Peripheral NKT Cells in Simian Immunodeficiency Virus-Infected Macaques

NKT cells are a specialized population of T lymphocytes that have an increasingly recognized role in immunoregulation, including controlling the response to viral infections. The characteristics of NKT cells in the peripheral blood of macaques during simian immunodeficiency virus (SIV) or chimeric simian/human immunodeficiency virus (HIV) (SHIV) infection were assessed. NKT cells comprised a mean of 0.19% of peripheral blood lymphocytes across the 64 uninfected macaques studied. Although the range in the percentages of NKT cells was large (0 to 2.2%), levels were stable over time within individual macaques without SIV/SHIV infection. The majority of NKT cells in macaques were CD4⁺ (on average 67%) with smaller populations being CD8⁺ (21%) and CD4/CD8 double positive (13%). A precipitous decline in CD4⁺ NKT cells occurred in all six macaques infected with CXCR4-tropic SHIV_mn229 early after infection, with a concomitant rise in CD8⁺ NKT cells in some animals. The depletion of CD4⁺ NKT cells was tightly correlated with the depletion of total CD4⁺ T cells. R5-tropic SIV_mac251 infection of macaques resulted in a slower and more variable decline in CD4⁺ NKT cells, with animals that were able to control SIV virus levels maintaining higher levels of CD4⁺ NKT cells. An inverse correlation between the depletion of total and CD4⁺ NKT cells and SIV viral load during chronic infection was observed. Our results demonstrate the infection-driven depletion of peripheral CD4⁺ NKT cells during both SHIV and SIV infection of macaques. Further studies of the implications of the loss of NKT cell subsets in the pathogenesis of HIV disease are needed.     

IgY antibodies against Ebola virus possess post-exposure protection in a murine pseudovirus challenge model and excellent thermostability

Ebola virus (EBOV) is one of the most virulent pathogens that causes hemorrhagic fever and displays high mortality rates and low prognosis rates in both humans and nonhuman primates. The post-exposure antibody therapies to prevent EBOV infection are considered effective as of yet. However, owing to the poor thermal stability of mammalian antibodies, their application in the tropics has remained limited. Therefore, a thermostable therapeutic antibody against EBOV was developed modelled on the poultry(chicken) immunoglobulin Y (IgY). The IgY antibodies retaining their neutralising activity at 25°C for one year, displayed excellent thermal stability, opposed to conventional polyclonal antibodies (pAbs) or monoclonal antibodies (mAbs). Laying hens were immunised with a variety of EBOV vaccine candidates and it was confirmed that VSVΔG/EBOVGP encoding the EBOV glycoprotein could induce high titer neutralising antibodies against EBOV. The therapeutic efficacy of immune IgY antibodies in vivo was evaluated in the newborn Balb/c mice who have been challenged with the VSVΔG/EBOVGP model. Mice that have been challenged with a lethal dose of the pseudovirus were treated 2 or 24 h post-infection with different doses of anti-EBOV IgY. The group receiving a high dose of 10⁶ NAU/kg (neutralising antibody units/kilogram) showed complete protection with no symptoms of a disease, while the low-dose group was only partially protected. Conversely, all mice receiving naive IgY died within 10 days. In conclusion, the anti-EBOV IgY exhibits excellent thermostability and protective efficacy. Anti-EBOV IgY shows a lot of promise in entering the realm of efficient Ebola virus treatment regimens.     

Immunogenic Profiling in Mice of a HIV/AIDS Vaccine Candidate (MVA-B) Expressing Four HIV-1 Antigens and Potentiation by Specific Gene Deletions

Background

The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B), that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs) with immunoregulatory function.

Methodology/Principal Findings

In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R), known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1β, respectively (referred as MVA-B ΔA41L/ΔB16R). A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B ΔA41L/ΔB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4⁺ and CD8⁺ T cells, with the CD8⁺ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B ΔA41L/ΔB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4⁺ and CD8⁺ T-cell immune responses. HIV-1-specific CD4⁺ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8⁺ T-cell responses, MVA-B ΔA41L/ΔB16R induced more GPN-specific CD8⁺ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env.

Conclusions/Significance

These findings revealed that MVA-B and MVA-B ΔA41L/ΔB16R induced in mice robust, polyfunctional and durable T-cell responses to HIV-1 antigens, but the double deletion mutant showed enhanced magnitude and quality of HIV-1 adaptive and memory responses. Our observations are relevant in the immune evaluation of MVA-B and on improvements of MVA vectors as HIV-1 vaccines.     

Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies

Background

Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines.

Principal Findings

We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg ⁻ recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2-fold) immunizations as compared to the udg ⁺ control virus MVA-gag, as determined by intracellular cytokine assay. In contrast, levels of HIV Gag-specific antibodies were elicited similarly in macaques following immunization with MVAΔudg-gag and MVA-gag. Furthermore, both udg ⁻ and udg ⁺ MVA vectors induced comparatively similar titers of MVA-specific neutralizing antibody responses following immunization of mice (over a 4-log range: 10⁴–10⁸ PFU) and rhesus macaques. These results suggest that the generation of MVA-specific neutralizing antibody responses are largely driven by input MVA antigens, rather than those that are synthesized de novo during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses.

Significance

Our identification of a spontaneously-immortalized (but not transformed) chicken embryo fibroblast cell line (DF-1) that is fully permissive for MVA growth and that can be engineered to stably express MVA genes provides the basis for a genetic system for MVA. DF-1 cells (and derivatives thereof) constitute viable alternatives, for the manufacture of MVA-based vaccines, to primary CEFs – the conventional cell substrate for MVA vaccines that is not amenable to genetic complementation strategies due to these cells'' finite lifespan in culture. The establishment of a genetic system for MVA, as illustrated here to allow udg deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors.     

Reduced protection from simian immunodeficiency virus SIVmac251 infection afforded by memory CD8+ T cells induced by vaccination during CD4+ T-cell deficiency

Adaptive CD4⁺ and CD8⁺ T-cell responses have been associated with control of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication. Here, we have designed a study with Indian rhesus macaques to more directly assess the role of CD8 SIV-specific responses in control of viral replication. Macaques were immunized with a DNA prime-modified vaccinia virus Ankara (MVA)-SIV boost regimen under normal conditions or under conditions of antibody-induced CD4⁺ T-cell deficiency. Depletion of CD4⁺ cells was performed in the immunized macaques at the peak of SIV-specific CD4⁺ T-cell responses following the DNA prime dose. A group of naïve macaques was also treated with the anti-CD4 depleting antibody as a control, and an additional group of macaques immunized under normal conditions was depleted of CD8⁺ T cells prior to challenge exposure to SIV_mac251. Analysis of the quality and quantity of vaccine-induced CD8⁺ T cells demonstrated that SIV-specific CD8⁺ T cells generated under conditions of CD4⁺ T-cell deficiency expressed low levels of Bcl-2 and interleukin-2 (IL-2), and plasma virus levels increased over time. Depletion of CD8⁺ T cells prior to challenge exposure abrogated vaccine-induced protection as previously shown. These data support the notion that adaptive CD4⁺ T cells are critical for the generation of effective CD8⁺ T-cell responses to SIV that, in turn, contribute to protection from AIDS. Importantly, they also suggest that long-term protection from disease will be afforded only by T-cell vaccines for HIV that provide a balanced induction of CD4⁺ and CD8⁺ T-cell responses and protect against early depletion of CD4⁺ T cells postinfection.     

Highly effective control of an AIDS virus challenge in macaques by using vesicular stomatitis virus and modified vaccinia virus Ankara vaccine vectors in a single-boost protocol

Previous studies have shown that vaccination and boosting of rhesus macaques with attenuated vesicular stomatitis virus (VSV) vectors encoding Env and Gag proteins of simian immunodeficiency virus-human immunodeficiency virus (SHIV) hybrid viruses protect rhesus macaques from AIDS after challenge with the highly pathogenic SHIV 89.6P (23). In the present study, we compared the effectiveness of a single prime-boost protocol consisting of VSV vectors expressing SHIV Env, Gag, and Pol proteins to that of a protocol consisting of a VSV vector prime followed with a single boost with modified vaccinia virus Ankara (MVA) expressing the same SHIV proteins. After challenge with SHIV 89.6P, MVA-boosted animals controlled peak challenge viral loads to less than 2 x 10(6) copies/ml (a level significantly lower than that seen with VSV-boosted animals and lower than those reported for other vaccine studies employing the same challenge). MVA-boosted animals have shown excellent preservation of CD4(+) T cells, while two of four VSV-boosted animals have shown significant loss of CD4(+) T cells. The improved protection in MVA-boosted animals correlates with trends toward stronger prechallenge CD8(+)-T-cell responses to SHIV antigens and stronger postchallenge SHIV-neutralizing antibody production.     

A single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector

Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.     

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.     

Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses

The search for a safe and efficacious vaccine for Ebola virus continues, as no current vaccine candidate is nearing licensure. We have developed (i) replication-competent, (ii) replication-deficient, and (iii) chemically inactivated rabies virus (RABV) vaccines expressing Zaire Ebola virus (ZEBOV) glycoprotein (GP) by a reverse genetics system based on the SAD B19 RABV wildlife vaccine. ZEBOV GP is efficiently expressed by these vaccine candidates and is incorporated into virions. The vaccine candidates were avirulent after inoculation of adult mice, and viruses with a deletion in the RABV glycoprotein had greatly reduced neurovirulence after intracerebral inoculation in suckling mice. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. The bivalent RABV/ZEBOV vaccines described here have several distinct advantages that may speed the development of inactivated vaccines for use in humans and potentially live or inactivated vaccines for use in nonhuman primates at risk of EBOV infection in endemic areas.     

CD8+ Lymphocytes Control Viral Replication in SIVmac239-Infected Rhesus Macaques without Decreasing the Lifespan of Productively Infected Cells

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.