The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017.

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analysed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunisation. This assessment allowed us to distinguish at least four antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

Two proteins, the hemagglutinin and the neuraminidase, protrude from the surface of the influenza virus. Their detection by the immune system allows the host organism to mount defences against the viral threat. The virus evolves in response to this pressure, which manifests as changes in the appearance of its hemagglutinin and neuraminidase. This process, known as antigenic drift, leads to the proteins evading detection. It is also why flu vaccines require frequent updates, as they rely on 'training' the immune system to recognise the most important strains in circulation ­ primarily by exposing it to appropriate versions of hemagglutinin. While the antigenic drift of hemagglutinin has been extensively studied, much less is known about how the neuraminidase accumulates mutations, and how these affect the immune response. To investigate this question, Catani et al. selected 43 genetically distant neuraminidases from human viral samples isolated between 2009 and 2017. Statistical analyses were applied to define their relatedness, revealing that a group of closely related neuraminidases predominated from 2009 to 2015, before they were being taken over by a second group. A third group, which was identified in viruses isolated in 2013, was remarkably close to the neuraminidase of strains that circulated in the late 1990s. The fourth and final group of neuraminidases was derived from influenza viruses that normally circulate in pigs but can also occasionally infect humans. Next, Catani et al. examined the immune response that these 43 neuraminidases could elicit in mice, as well as in ferrets ­ the animal most traditionally used in influenza research. This allowed them to pinpoint which changes in the neuraminidase sequences were important to escape recognition by the host. Data obtained from the two model species were comparable, suggesting that these experiments could be conducted on mice going forward, which are easier to work with than ferrets. Finally, Catani et al. used machine learning to build a computational model that could predict how strongly the immune system would respond to a specific neuraminidase variant. These findings could help guide the development of new vaccines that include neuraminidases tailored to best prime and train the immune system against a larger variety of strains. This may aid the development of 'supra-seasonal' vaccines that protect against a broad range of influenza viruses, reducing the need for yearly updates.

Anti-neuraminidase and anti-hemagglutinin immune serum can confer inter-lineage cross protection against recent influenza B.

Influenza B viruses (IBV) are responsible for a considerable part of the burden caused by influenza virus infections. Since their emergence in the 1980s, the Yamagata and Victoria antigenic lineages of influenza B circulate in alternate patterns across the globe. Furthermore, their evolutionary divergence and the appearance of new IBV subclades complicates the prediction of future influenza vaccines compositions. It has been proposed that the addition of the neuraminidase (NA) antigen could potentially induce a broader protection and compensate for hemagglutinin (HA) mismatches in the current vaccines. Here we show that anti-NA and -HA sera against both Victoria and Yamagata lineages have limited inter-lineage cross-reactivity. When transferred to mice prior to infection with a panel of IBVs, anti-NA sera were as potent as anti-HA sera in conferring protection against homologous challenge and, in some cases, conferred superior protection against challenge with heterologous IBV strains.

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

Pre-existing antibodies directed against a tetramerizing domain enhance the immune response against artificially stabilized soluble tetrameric influenza neuraminidase.

The neuraminidase (NA) is an abundant antigen at the surface of influenza virions. Recent studies have highlighted the immune-protective potential of NA against influenza and defined anti-NA antibodies as an independent correlate of protection. Even though NA head domain changes at a slightly slower pace than hemagglutinin (HA), NA is still subject to antigenic drift, and therefore an NA-based influenza vaccine antigen may have to be updated regularly and thus repeatedly administered. NA is a tetrameric type II membrane protein, which readily dissociates into dimers and monomers when expressed in a soluble form. By using a tetramerizing zipper, such as the tetrabrachion (TB) from Staphylothermus marinus, it is possible to stabilize soluble NA in its active tetrameric conformation, an imperative for the optimal induction of protective NA inhibitory antibodies. The impact of repetitive immunizations with TB-stabilized antigens on the immunogenicity of soluble TB-stabilized NA is unknown. We demonstrate that TB is immunogenic in mice. Interestingly, preexisting anti-TB antibodies enhance the anti-NA antibody response induced by immunization with TB-stabilized NA. This immune-enhancing effect was transferable by serum and operated independently of activating Fcγ receptors. We also demonstrate that priming with TB-stabilized NA antigens, enhances the NA inhibitory antibody responses against a heterosubtypic TB-stabilized NA. These findings have implications for the clinical development of oligomeric vaccine antigens that are stabilized by a heterologous oligomerizing domain.

Mapping of a Novel H3-Specific Broadly Neutralizing Monoclonal Antibody Targeting the Hemagglutinin Globular Head Isolated from an Elite Influenza Virus-Immunized Donor Exhibiting Serological Breadth.

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design.IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design.

Comparison of adjuvants to optimize influenza neutralizing antibody responses.

Seasonal influenza vaccines represent a positive intervention to limit the spread of the virus and protect public health. Yet continual influenza evolution and its ability to evade immunity pose a constant threat. For these reasons, vaccines with improved potency and breadth of protection remain an important need. We previously developed a next-generation influenza vaccine that displays the trimeric influenza hemagglutinin (HA) on a ferritin nanoparticle (NP) to optimize its presentation. Similar to other vaccines, HA-nanoparticle vaccine efficacy is increased by the inclusion of adjuvants during immunization. To identify the optimal adjuvants to enhance influenza immunity, we systematically analyzed TLR agonists for their ability to elicit immune responses. HA-NPs were compatible with nearly all adjuvants tested, including TLR2, TLR4, TLR7/8, and TLR9 agonists, squalene oil-in-water mixtures, and STING agonists. In addition, we chemically conjugated TLR7/8 and TLR9 ligands directly to the HA-ferritin nanoparticle. These TLR agonist-conjugated nanoparticles induced stronger antibody responses than nanoparticles alone, which allowed the use of a 5000-fold-lower dose of adjuvant than traditional admixtures. One candidate, the oil-in-water adjuvant AF03, was also tested in non-human primates and showed strong induction of neutralizing responses against both matched and heterologous H1N1 viruses. These data suggest that AF03, along with certain TLR agonists, enhance strong neutralizing antibody responses following influenza vaccination and may improve the breadth, potency, and ultimately vaccine protection in humans.

Development of a Pan-H1 Influenza Vaccine.

The efficacy of current seasonal influenza vaccines varies greatly, depending on the match to circulating viruses. Although most vaccines elicit strain-specific responses, some present cross-reactive epitopes that elicit antibodies against diverse viruses and remain unchanged and effective for several years. To determine whether combinations of specific H1 hemagglutinin (HA) antigens stimulate immune responses that protect against diverse H1 influenza viruses, we evaluated the antibody responses elicited by HA-ferritin nanoparticles derived from six evolutionarily divergent H1 sequences and two computationally optimized broadly reactive antigen (COBRA) HA antigens. Humoral responses were assessed against a panel of 16 representative influenza virus strains from the past 80 years. HAs from the strains A/NewCaledonia/20/1999 (NC99), A/California/04/2009 (CA09), A/HongKong/117/1977 (HK77), COBRA X6, or P1 elicited neutralization against diverse strains, and a combination of three wild-type HA or two COBRA HA nanoparticles conferred significant additional breadth beyond that observed with any individual strain. Therefore, combinations of H1 HAs may constitute a pan-H1 influenza vaccine.IMPORTANCE Seasonal influenza vaccines elicit strain-specific immune responses designed to protect against circulating viruses. Because these vaccines often show limited efficacy, the search for a broadly protective seasonal vaccine remains a priority. Among different influenza virus subtypes, H1N1 has long been circulating in humans and has caused pandemic outbreaks. In order to assess the potential of a multivalent HA combination vaccine to improve the breadth of protection against divergent H1N1 viruses, HA-ferritin nanoparticles were made and evaluated in mice against a panel of historical and contemporary influenza virus strains. Trivalent combinations of H1 nanoparticles improved the breadth of immunity against divergent H1 influenza viruses.

Impact of age and pre-existing influenza immune responses in humans receiving split inactivated influenza vaccine on the induction of the breadth of antibodies to influenza A strains.

Most humans have pre-existing immunity to influenza viruses. In this study, volunteers (ages of 18-85 years) were vaccinated with split, inactivated Fluzone™ influenza vaccine in four consecutive influenza seasons from 2013 to 2016 seasons. The impact of repeated vaccination on breadth and durability of antibodies was assessed as a result of vaccine strain changes. Total IgG anti-hemagglutinin (HA) binding antibodies and hemagglutination-inhibition (HAI) activity increased in all age groups against both influenza A HA components in the vaccine post-vaccination (day 21). However, younger subjects maintained seroprotective titers to the vaccine strains, which resulted in higher seroconversion rates in the elderly, since the HAI titers in elderly subjects were more likely to decline prior to the next season. Young subjects had significant HAI activity against historical, as well as contemporary H1 and H3 vaccine strains from the mid-1980s to present. In contrast, elderly subjects had HAI activity to H1 strains from all years, but were more likely to have HAI activity to older strains from 1918-1950s. They also had a more restricted HAI profile against H3 viruses compared to young subjects recognizing H3N2 influenza viruses from the mid-2000s to present. Vaccine recipients were then categorized by whether subjects seroconverted from a seronegative or seropositive pre-vaccination state. Regardless of age, immunological recall or 'back-boosting' to antigenically related strains were associated with seroconversion to the vaccine strain. Overall, both younger and older people have the ability to mount a breadth of immune responses following influenza vaccination. This report describes how imprinting exposure differs across age groups, influences antibody cross-reactivity to past hemagglutinin antigenic variants, and shapes immune responses elicited by current split inactivated influenza vaccines. Understanding how current influenza vaccines are influenced by pre-existing immunity in people of different ages is critical for designing the next-generation of 'universal' or broadly-protective influenza vaccines.

Elicitation of Protective Antibodies against a Broad Panel of H1N1 Viruses in Ferrets Preimmune to Historical H1N1 Influenza Viruses.

Most preclinical animal studies test influenza vaccines in immunologically naive animal models, even though the results of vaccination may not accurately reflect the effectiveness of vaccine candidates in humans that have preexisting immunity to influenza. In this study, novel, broadly reactive influenza vaccine candidates were assessed in preimmune ferrets. These animals were infected with different H1N1 isolates before being vaccinated or infected with another influenza virus. Previously, our group has described the design and characterization of computationally optimized broadly reactive hemagglutinin (HA) antigens (COBRA) for H1N1 isolates. Vaccinating ferrets with virus-like particle (VLP) vaccines expressing COBRA HA proteins elicited antibodies with hemagglutination inhibition (HAI) activity against more H1N1 viruses in the panel than VLP vaccines expressing wild-type HA proteins. Specifically, ferrets infected with the 1986 virus and vaccinated with a single dose of the COBRA HA VLP vaccines elicited antibodies with HAI activity against 11 to 14 of the 15 H1N1 viruses isolated between 1934 and 2013. A subset of ferrets was infected with influenza viruses expressing the COBRA HA antigens. These COBRA preimmune ferrets had superior breadth of HAI activity after vaccination with COBRA HA VLP vaccines than COBRA preimmune ferrets vaccinated with VLP vaccines expressing wild-type HA proteins. Overall, priming naive ferrets with COBRA HA based viruses or using COBRA HA based vaccines to boost preexisting antibodies induced by wild-type H1N1 viruses, COBRA HA antigens elicited sera with the broadest HAI reactivity against multiple antigenic H1N1 viral variants. This is the first report demonstrating the effectiveness of a broadly reactive or universal influenza vaccine in a preimmune ferret model.IMPORTANCE Currently, many groups are testing influenza vaccine candidates to meet the challenge of developing a vaccine that elicits broadly reactive and long-lasting protective immune responses. The goal of these vaccines is to stimulate immune responses that react against most, if not all, circulating influenza strains, over a long period of time in all populations of people. Commonly, these experimental vaccines are tested in naive animal models that do not have anti-influenza immune responses; however, humans have preexisting immunity to influenza viral antigens, particularly antibodies to the HA and NA glycoproteins. Therefore, this study investigated how preexisting antibodies to historical influenza viruses influenced HAI-specific antibodies and protective efficacy using a broadly protective vaccine candidate.

Application of replication-defective West Nile virus vector to non-flavivirus vaccine targets.

The RepliVax vaccine platform(RV) is based on flavivirus genomes that are rationally attenuated by deletion. The self-limiting infection provided by RV has been demonstrated to be safe, highly immunogenic and efficacious for several vaccine candidates against flaviviruses. Here respiratory syncytial virus (RSV) F, influenza virus HA, and simian immunodeficiency virus (SIV) Env proteins were expressed in place of either prM-E or C-prM-E gene deletions of the West Nile (WN) virus genome. The resulting RV-RSV, -influenza and -SIV vaccine prototypes replicated efficiently in complementing helper cells expressing the WN structural proteins in trans. Expressed antigens exhibited correct post-translational processing and the RV recombinants were shown to be highly attenuated and immunogenic in mice, eliciting strong antigen-specific antibodies as well as detectable T-cell responses. These data support the utility of RV vectors for development of vaccines against non-flavivirus targets including rabies and HIV.

Long-Lasting Cross-Protection Against Influenza A by Neuraminidase and M2e-based immunization strategies.

There is mounting evidence that in the absence of neutralizing antibodies cross-reactive T cells provide protection against pandemic influenza viruses. Here, we compared protection and CD8+ T cell responses following challenge with H1N1 2009 pandemic and H3N2 viruses of mice that had been immunized with hemagglutinin (HA), neuraminidase (NA) and the extracellular domain of matrix protein 2 (M2e) fused to a virus-like particle (VLP). Mice were challenged a first time with a sublethal dose of H1N1 2009 pandemic virus and, four weeks later, challenged again with an H3N2 virus. Mice that had been vaccinated with HA, NA, NA + M2e-VLP and HA + NA + M2e-VLP were protected against homologous H1N1 virus challenge. Challenged NA and NA + M2e-VLP vaccinated mice mounted CD8+ T cell responses that correlated with protection against secondary H3N2 challenge. HA-vaccinated mice were fully protected against challenge with homologous H1N1 2009 virus, failed to mount cross-reactive CD8+ T cells and succumbed to the second challenge with heterologous H3N2 virus. In summary, NA- and M2e-based immunity can protect against challenge with (homologous) virus without compromising the induction of robust cross-reactive CD8+ T cell responses upon exposure to virus.

Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses.

UNLABELLED: One of the challenges of developing influenza A vaccines is the diversity of antigenically distinct isolates. Previously, a novel hemagglutinin (HA) for H5N1 influenza was derived from a methodology termed computationally optimized broadly reactive antigen (COBRA). This COBRA HA elicited a broad antibody response against H5N1 isolates from different clades. We now report the development and characterization of a COBRA-based vaccine for both seasonal and pandemic H1N1 influenza virus isolates. Nine prototype H1N1 COBRA HA proteins were developed and tested in mice using a virus-like particle (VLP) format for the elicitation of broadly reactive, functional antibody responses and protection against viral challenge. These candidates were designed to recognize H1N1 viruses isolated within the last 30 years. In addition, several COBRA candidates were designed based on sequences of H1N1 viruses spanning the past 100 years, including modern pandemic H1N1 isolates. Four of the 9 H1N1 COBRA HA proteins (X1, X3, X6, and P1) had the broadest hemagglutination inhibition (HAI) activity against a panel of 17 H1N1 viruses. These vaccines were used in cocktails or prime-boost combinations. The most effective regimens that both elicited the broadest HAI response and protected mice against a pandemic H1N1 challenge were vaccines that contained the P1 COBRA VLP and either the X3 or X6 COBRA VLP vaccine. These mice had little or no detectable viral replication, comparable to that observed with a matched licensed vaccine. This is the first report describing a COBRA-based HA vaccine strategy that elicits a universal, broadly reactive, protective response against seasonal and pandemic H1N1 isolates. IMPORTANCE: Universal influenza vaccine approaches have the potential to be paradigm shifting for the influenza vaccine field, with the goal of replacing the current standard of care with broadly cross-protective vaccines. We have used COBRA technology to develop an HA head-based strategy that elicits antibodies against many H1 strains that have undergone genetic drift and has potential as a "subtype universal" vaccine. Nine HA COBRA candidates were developed, and these vaccines were used alone, in cocktails or in prime-boost combinations. The most effective regimens elicited the broadest hemagglutination inhibition (HAI) response against a panel of H1N1 viruses isolated over the past 100 years. This is the first report describing a COBRA-based HA vaccine strategy that elicits a broadly reactive response against seasonal and pandemic H1N1 isolates.

Murine responses to recombinant MVA versus ALVAC vaccines against tumor-associated antigens, gp100 and 5T4.

Virally vectored cancer vaccines comprise a new form of immunotherapy that aim to generate anti-tumor immune responses with potential for tumor clearance and enhanced patient survival. Here, we compared 2 replication-deficient poxviruses modified vaccinia Ankara (MVA) and ALVAC(2) in their ability to induce antigen expression and immunogenicity of the tumor-associated antigens (TAAs) 5T4 and gp100. To facilitate the comparison, recombinant MVA-gp100M and ALVAC(2)-5T4 were constructed to complement existing ALVAC(2)-gp100M and MVA-5T4 vectors. Recombinant TAA expression in chicken embryo fibroblast cells was confirmed by Western blot analysis. 5T4 expression was approximately equal for both viruses, whereas ALVAC-derived gp100 was quickly degraded, at a time point when MVA-derived gp100 was still stable and expressed at high levels. Human leukocyte antigen-A2 transgenic mice were vaccinated with recombinant viruses and the CD8 T-cell responses elicited against each TAA were monitored by interferon-γ enzyme-linked immunospot. No 5T4 peptide responses were detected using splenocytes from mice vaccinated with either vector, whereas vaccination with MVA elicited a significantly higher gp100-specific response than ALVAC(2) at 10 PFU (P<0.001). In CD-1 mice, each vector elicited similar 5T4 antibody responses, whereas MVA was more potent and induced gp100 antibody responses at a lower immunization dose than ALVAC (P<0.001). In this study, immunogenicity varied depending on the viral vector used and reflected vector-associated differences in in vitro TAA expression and stability. These findings suggest that novel vector-transgene combinations must be assessed individually when designing vaccines, and that stability of vector-encoded proteins produced in vitro may be useful as a predictor for in vitro immunogenicity.

Preclinical qualification of a new multi-antigen candidate vaccine for metastatic melanoma.

New therapies are urgently required for the treatment of patients with melanoma. Here we describe the generation and preclinical evaluation of 3 new recombinant ALVAC(2) poxviruses vCP2264, vCP2291, and vCP2292 for their ability to induce the desired cellular immune responses against the encoded melanoma-associated antigens. This was done either in HLA-A2/K transgenic mice or using in vitro antigen-presentation studies. These studies demonstrated that the vaccine was able to induce HLA-A*0201-restricted T-cell responses against gp100 and NY-ESO-1, detectable directly ex vivo, in HLA-A2/K-transgenic mice. The in vitro antigen presentation studies, in the absence of appropriate animal models, demonstrated that target cells infected with the vaccine construct were lysed by MAGE-1, MAGE-3 or MART-1 peptide-specific T cells. These data indicate that ALVAC(2)-encoded melanoma-associated antigens can be properly processed and presented to induce antigen-specific cytotoxic T-cell responses. To enhance the immunogenicity of the melanoma antigens, a TRIad of COstimulatory Molecules (TRICOM) were also cloned into all 3 vectors. Increased in vitro proliferation and IFN-γ production was observed with all ALVAC(2) poxviruses encoding TRICOM, confirming the immune-enhancing effect of the ALVAC-encoded TRICOM. These studies demonstrated that all components of the vaccine were functionally active and provide a rationale for moving this candidate vaccine to the clinic.

Multispecific vaccine-induced mucosal cytotoxic T lymphocytes reduce acute-phase viral replication but fail in long-term control of simian immunodeficiency virus SIVmac239.

Given the current difficulties generating vaccine-induced neutralizing antibodies to human immunodeficiency virus (HIV), the focus of the vaccine community has shifted toward creating cytotoxic-T-lymphocyte (CTL)-based vaccines. Recent reports of CTL-based vaccine trials in macaques challenged with simian/human immunodeficiency virus SHIV-89.6P have supported the notion that such vaccines can ameliorate the course of disease. However, almost all of these studies included Env as an immunogen and since SHIV-89.6P is sensitive to neutralizing antibodies it is difficult to determine the mechanism(s) of protection. Consequently, SHIV-89.6P challenge of macaques may be a poor model for determining vaccine efficacy in humans. To ascertain the effect of vaccine-induced multispecific mucosal CTL, in the absence of Env-specific antibody, on the control of an immunodeficiency virus challenge, we vaccinated Mamu-A*01(+) macaques with constructs encoding a combination of CTL epitopes and full-length proteins (Tat, Rev, and Nef) by using a DNA prime/recombinant modified vaccinia virus Ankara (rMVA) boost regimen. The vaccination induced virus-specific CTL and CD4(+) helper T lymphocytes with CTL frequencies as high as 20,000/million peripheral blood mononuclear cells. The final rMVA vaccination, delivered intravenously, engendered long-lived mucosal CTL. At 16 weeks after the final rMVA vaccination, the vaccinees and naive, Mamu-A*01(+) controls were challenged intrarectally with SIVmac239. Massive early anamnestic cellular immune responses controlled acute-phase viral replication; however, the three vaccinees were unable to control virus replication in the chronic phase. The present study suggests that multispecific mucosal CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic-phase viral replication after a high-dose mucosal challenge with a pathogenic simian immunodeficiency virus.

Differences between T cell epitopes recognized after immunization and after infection.

Evidence suggests that cellular immune responses play a crucial role in the control of HIV and SIV replication in infected individuals. Several vaccine strategies have therefore targeted these CD8(+) and CD4(+) responses. Whether vaccination induces the same repertoire of responses seen after infection is, however, a key unanswered question in HIV vaccine development. We therefore compared the epitope specificity induced by vaccination to that present postchallenge in the peripheral blood. Intracellular cytokine staining of PBMC stimulated with overlapping 15/20-mer peptides spanning the proteins of SIV were measured after DNA/modified vaccinia Ankara vaccination of eight rhesus macaques. Lymphocytes from 8 animals recognized a total of 39 CD8 epitopes and 41 CD4 epitopes encoded by the vaccine. T cell responses were again monitored after challenge with SIVmac239 to investigate the evolution of these responses. Only 57% of all CD8(+) T cell responses and 19% of all CD4(+) T cell responses present after vaccination were recalled after infection as measured in the peripheral blood. Interestingly, 29 new CD8 epitopes and 5 new CD4 epitopes were recognized by PBMC in the acute phase. These new epitopes were not detected after vaccination, and only some of them were maintained in the chronic phase (33% of CD8 and no CD4 responses). Additionally, 24 new CD8 epitopes and 7 new CD4 epitopes were recognized by PBMC in the chronic phase of infection. The repertoire of the immune response detected in the peripheral blood after immunization substantially differed from the immune response detected in the peripheral blood after infection.

Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule, Mamu-A*02: a paradigm for virus evolution and persistence?

It is now accepted that an effective vaccine against AIDS must include effective cytotoxic-T-lymphocyte (CTL) responses. The simian immunodeficiency virus (SIV)-infected rhesus macaque is the best available animal model for AIDS, but analysis of macaque CTL responses has hitherto focused mainly on epitopes bound by a single major histocompatibility complex (MHC) class I molecule, Mamu-A*01. The availability of Mamu-A*01-positive macaques for vaccine studies is therefore severely limited. Furthermore, it is becoming clear that different CTL responses are able to control immunodeficiency virus replication with varying success, making it a priority to identify and analyze CTL responses restricted by common MHC class I molecules other than Mamu-A*01. Here we describe two novel epitopes derived from SIV, one from Gag (Gag(71-79) GY9), and one from the Nef protein (Nef(159-167) YY9). Both epitopes are bound by the common macaque MHC class I molecule, Mamu-A*02. The sequences of these two eptiopes are consistent with the molecule's peptide-binding motif, which we have defined by elution of natural ligands from Mamu-A*02. Strikingly, we found evidence for the selection of escape variant viruses by CTL specific for Nef(159-167) YY9 in 6 of 6 Mamu-A*02-positive animals. In contrast, viral sequences encoding the Gag(71-79) GY9 epitope remained intact in each animal. This situation is reminiscent of Mamu-A*01-restricted CTL that recognize Tat(28-35) SL8, which reproducibly selects for escape variants during acute infection, and Gag(181-189) CM9, which does not. Differential selection by CTL may therefore be a paradigm of immunodeficiency virus infection.

Immunization of rhesus macaques with a DNA prime/modified vaccinia virus Ankara boost regimen induces broad simian immunodeficiency virus (SIV)-specific T-cell responses and reduces initial viral replication but does not prevent disease progression following challenge with pathogenic SIVmac239.

Producing a prophylactic vaccine for human immunodeficiency virus (HIV) has proven to be a challenge. Most biological isolates of HIV are difficult to neutralize, so that conventional subunit-based antibody-inducing vaccines are unlikely to be very effective. In the rhesus macaque model, some protection was afforded by DNA/recombinant viral vector vaccines. However, these studies used as the challenge virus SHIV-89.6P, which is neutralizable, making it difficult to determine whether the observed protection was due to cellular immunity, humoral immunity, or a combination of both. In this study, we used a DNA prime/modified vaccinia virus Ankara boost regimen to immunize rhesus macaques against nearly all simian immunodeficiency virus (SIV) proteins. These animals were challenged intrarectally with pathogenic molecularly cloned SIVmac239, which is resistant to neutralization. The immunization regimen resulted in the induction of virus-specific CD8(+) and CD4(+) responses in all vaccinees. Although anamnestic neutralizing antibody responses against laboratory-adapted SIVmac251 developed after the challenge, no neutralizing antibodies against SIVmac239 were detectable. Vaccinated animals had significantly reduced peak viremia compared with controls (P < 0.01). However, despite the induction of virus-specific cellular immune responses and reduced peak viral loads, most animals still suffered from gradual CD4 depletion and progressed to disease.