A new theory of cytotoxic T-lymphocyte memory: implications for HIV treatment

We use simple mathematical models to examine the dynamics of primary and secondary cytotoxic T-lymphocyte (CTL) responses to viral infections. In particular, we are interested in conditions required to resolve the infection and to protect the host upon secondary challenge. While protection against reinfection is only effective in a restricted set of circumstances, we find that resolution of the primary infection requires persistence of CTL precursors (GTLp), as well as a fast rate of activation of the CTLp. Since these are commonly the defining characteristics of CTL memory, we propose that CTL memory may have evolved in order to clear the virus during primary challenge. We show experimental data from lymphocytic choriomeningitis virus infection in mice, supporting our theory on CTL memory. We adapt our models to HIV and find that immune impairment during the primary phase of the infection may result in the failure to establish CTL memory which in turn leads to viral persistence. Based on our models we suggest conceptual treatment regimes which ensure establishment of CTL memory. This would allow the immune response to control HIV in the long term in the absence of continued therapy.     

Systemic immunity and mucosal immunity are induced against human immunodeficiency virus Gag protein in mice by a new hyperattenuated strain of Listeria monocytogenes

Vaccines designed to control chronic infections by intracellular agents such as human immunodeficiency virus type 1 (HIV-1) require the induction of cell-mediated immune responses to rid the host of pathogen-infected cells. Listeria monocytogenes has characteristics that make it an attractive vaccine vector for use against such infections. Here we show that parenteral immunization with a new highly attenuated strain of this organism provided complete protection against systemic and mucosal challenges with a recombinant vaccinia virus expressing HIV-1 gag. Immunization also generated a strong, long-term memory cytotoxic-T-lymphocyte (CTL) response in spleen, mesenteric lymph nodes, and Peyer's patches directed against the gag protein. Oral immunization with this attenuated strain also produced complete, long-lasting protection against the recombinant virus but only against mucosal virus challenge. Curiously, oral immunization was associated with a transient CTL response in the three lymphoid tissues examined.     

Protection against CTL escape and clinical disease in a murine model of virus persistence

CTL escape mutations have been identified in several chronic infections, including mice infected with mouse hepatitis virus strain JHM. One outstanding question in understanding CTL escape is whether a CD8 T cell response to two or more immunodominant CTL epitopes would prevent CTL escape. Although CTL escape at multiple epitopes seems intuitively unlikely, CTL escape at multiple CD8 T cell epitopes has been documented in some chronically infected individual animals. To resolve this apparent contradiction, we engineered a recombinant variant of JHM that expressed the well-characterized gp33 epitope of lymphocytic choriomeningitis virus, an epitope with high functional avidity. The results show that the presence of a host response to this second epitope protected mice against CTL escape at the immunodominant JHM-specific CD8 T cell epitope, the persistence of infectious virus, and the development of clinical disease.     

Vaccinia virus-specific human CD4+ cytotoxic T-lymphocyte clones.

Vaccinia virus-specific cytotoxic T-lymphocyte (CTL) clones were established from a healthy donor, who had been immunized with vaccinia virus vaccine, by stimulation of peripheral blood lymphocytes with UV-inactivated vaccinia virus antigen. The phenotype of all of the clones established was CD3+ CD4+ CD8- Leu11-. We used a panel of allogenic vaccinia virus-infected B-lymphoblastoid cell lines and demonstrated that some of the clones recognized vaccinia virus epitopes presented by human leukocyte antigen (HLA) class II molecules. Monoclonal antibodies specific for either HLA-DP or HLA-DR determinant reduced the cytotoxicity of specific clones. The HLA-restricted cytotoxicity of the clones is vaccinia virus specific, because vaccinia virus-infected but not influenza virus-infected autologous target cells were lysed. Using vaccinia virus deletion mutants, we found that some of the CTL clones recognize an epitope(s) that lies within the HindIII KF regions of the vaccinia virus genome. These results indicate that heterogeneous CD4+ CTL clones specific for vaccinia virus are induced in response to infection and may be important in recovery from and protection against poxvirus infections.     

Correlates of cytotoxic T-lymphocyte-mediated virus control: implications for immunosuppressive infections and their treatment

A very important question in immunology is to determine which factors decide whether an immune response can efficiently clear or control a viral infection, and under what circumstances we observe persistent viral replication and pathology. This paper summarizes how mathematical models help us gain new insights into these questions, and explores the relationship between antiviral therapy and long-term immunological control in human immunodeficiency virus (HIV) infection. We find that cytotoxic T lymphocyte (CTL) memory, defined as antigen-independent persistence of CTL precursors, is necessary for the CTL response to clear an infection. The presence of such a memory response is associated with the coexistence of many CTL clones directed against multiple epitopes. If CTL memory is inefficient, then persistent replication can be established. This outcome is associated with a narrow CTL response directed against only one or a few viral epitopes. If the virus replicates persistently, occurrence of pathology depends on the level of virus load at equilibrium, and this can be determined by the overall efficacy of the CTL response. Mathematical models suggest that controlled replication is reflected by a positive correlation between CTLs and virus load. On the other hand, uncontrolled viral replication results in higher loads and the absence of a correlation between CTLs and virus load. A negative correlation between CTLs and virus load indicates that the virus actively impairs immunity, as observed with HIV. Mathematical models and experimental data suggest that HIV persistence and pathology are caused by the absence of sufficient CTL memory. We show how mathematical models can help us devise therapy regimens that can restore CTL memory in HIV patients and result in long-term immunological control of the virus in the absence of life-long treatment.     

Immunogenicity of a Human Immunodeficiency Virus (HIV) Polytope Vaccine Containing Multiple HLA A2 HIV CD8+ Cytotoxic T-Cell Epitopes

Compelling evidence now suggests that alphabeta CD8 cytotoxic T lymphocytes (CTL) have an important role in preventing human immunodeficiency virus (HIV) infection and/or slowing progression to AIDS. Here, we describe an HIV type 1 CTL polyepitope, or polytope, vaccine comprising seven contiguous minimal HLA A2-restricted CD8 CTL epitopes conjoined in a single artificial construct. Epitope-specific CTL lines derived from HIV-infected individuals were able to recognize every epitope within the construct, and HLA A2-transgenic mice immunized with a recombinant virus vaccine coding for the HIV polytope also generated CTL specific for different epitopes. Each epitope in the polytope construct was therefore processed and presented, illustrating the feasibility of the polytope approach for HIV vaccine design. By simultaneously inducing CTL specific for different epitopes, an HIV polytope vaccine might generate activity against multiple challenge isolates and/or preempt the formation of CTL escape mutants.     

Modelling the dynamics of LCMV infection in mice: II. Compartmental structure and immunopathology

In this study, we develop a mathematical model for analysis of the compartmental aspects and immunopathology of lymphocytic choriomeningitis virus (LCMV) infection in mice. We used sets of original and published data on systemic (extrasplenic) virus distribution to estimate the parameters of virus growth and elimination for spleen and other anatomical compartments, such as the liver, kidney, thymus and lung as well as transfer rates between blood and the above organs. A mathematical model quantitatively integrating the virus distribution kinetics in the host, the specific cytotoxic T lymphocyte (CTL) response in spleen and the re-circulation of effector CTL between spleen, blood and liver is advanced to describe the CTL-mediated immunopathology (hepatitis) in mice infected with LCMV. For intravenous and "peripheral" routes of infection we examine the severity of the liver disease, as a function of the virus dose and the host's immune status characterized by the numbers of precursor and/or cytolytic effector CTL. The model is used to predict the efficacy of protection against virus persistence and disease in a localized viral infection as a function of the composition of CTL population. The modelling analysis suggests quantitative demands to CTL memory for maximal protection against a wide range of doses of infection with a primarily peripheral site of virus replication without the risk of favoring immunopathology. It specifies objectives for CTL vaccination to ensure virus elimination with minimal immunopathology vs. vaccination for disease.     

Kinetics of the Development of Protective Immunity in Mice Vaccinated with a Live Attenuated Retrovirus

Vaccination of mice with a live attenuated vaccine virus induces potent protection against subsequent challenge with pathogenic Friend retroviral complex. The kinetic studies presented here demonstrate protection from acute splenomegaly as early as 1 week postvaccination. At this time point virus-specific cytotoxic T lymphocytes (CTL) were demonstrable in direct chromium release assays. However, during the first 2 weeks after vaccination protection was incomplete since the mice were not protected against establishment of low-level persistent infections in the spleen. By 3 weeks postvaccination the animals were protected against the establishment of persistent virus as well as acute splenomegaly. The timing of this complete protection correlated with the presence of both virus-neutralizing antibodies and primed CTL in the immunized mice. Within 3 days of virus challenge, vaccinated mice showed high levels of activated B cells and CD4(+) and CD8(+) T cells, indicating an efficient priming of all lymphocyte subsets. Despite very limited replication of the vaccine virus, the protective effect was long lived and was still present 6 months after immunization.     

Adaptation to human populations is revealed by within-host polymorphisms in HIV-1 and hepatitis C virus

CD8(+) cytotoxic T-lymphocytes (CTLs) perform a critical role in the immune control of viral infections, including those caused by human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). As a result, genetic variation at CTL epitopes is strongly influenced by host-specific selection for either escape from the immune response, or reversion due to the replicative costs of escape mutations in the absence of CTL recognition. Under strong CTL-mediated selection, codon positions within epitopes may immediately "toggle" in response to each host, such that genetic variation in the circulating virus population is shaped by rapid adaptation to immune variation in the host population. However, this hypothesis neglects the substantial genetic variation that accumulates in virus populations within hosts. Here, we evaluate this quantity for a large number of HIV-1- (n > or = 3,000) and HCV-infected patients (n > or = 2,600) by screening bulk RT-PCR sequences for sequencing "mixtures" (i.e., ambiguous nucleotides), which act as site-specific markers of genetic variation within each host. We find that nonsynonymous mixtures are abundant and significantly associated with codon positions under host-specific CTL selection, which should deplete within-host variation by driving the fixation of the favored variant. Using a simple model, we demonstrate that this apparently contradictory outcome can be explained by the transmission of unfavorable variants to new hosts before they are removed by selection, which occurs more frequently when selection and transmission occur on similar time scales. Consequently, the circulating virus population is shaped by the transmission rate and the disparity in selection intensities for escape or reversion as much as it is shaped by the immune diversity of the host population, with potentially serious implications for vaccine design.     

Identification of Cytotoxic T Lymphocyte Epitopes on Swine Viruses: Multi-Epitope Design for Universal T Cell Vaccine

Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8⁺ T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES.     

A polytope DNA vaccine elicits multiple effector and memory CTL responses and protects against human papillomavirus 16 E7-expressing tumour

Vaccine-induced CD8 T cells directed to tumour-specific antigens are recognised as important components of protective and therapeutic immunity against tumours. Where tumour antigens have pathogenic potential or where immunogenic epitopes are lost from tumours, development of subunit vaccines consisting of multiple individual epitopes is an attractive alternative to immunising with whole tumour antigen. In the present study we investigate the efficacy of two DNA-based multiepitope (polytope) vaccines containing murine (H-2^b) and human (HLA-A*0201)–restricted epitopes of the E7 oncoprotein of human papillomavirus type 16, in eliciting tumour-protective cytotoxic T-lymphocyte (CTL) responses. We show that the first of these polytopes elicited powerful effector CTL responses (measured by IFN- ELISpot) and long-lived memory CTL responses (measured by functional CTL assay and tetramers) in immunised mice. The responses could be boosted by immunisation with a recombinant vaccinia virus expressing the polytope. Responses induced by immunisation with polytope DNA alone partially protected against infection with recombinant vaccinia virus expressing the polytope. Complete protection was afforded against challenge with an E7-expressing tumour, and reduced growth of nascent tumours was observed. A second polytope differing in the exact composition and order of CTL epitopes, and lacking an inserted endoplasmic reticulum targeting sequence and T-helper epitope, induced much poorer CTL responses and failed to protect against tumour challenge. These observations indicate the validity of a DNA polytope vaccine approach to human papillomavirus E7–associated carcinoma, and underscore the importance of design in polytope vaccine construction.     

Dynamics of immune escape during HIV/SIV infection

Several studies have shown that cytotoxic T lymphocytes (CTLs) play an important role in controlling HIV/SIV infection. Notably, the observation of escape mutants suggests a selective pressure induced by the CTL response. However, it remains difficult to assess the definite role of the cellular immune response. We devise a computational model of HIV/SIV infection having a broad cellular immune response targeting different viral epitopes. The CTL clones are stimulated by viral antigen and interact with the virus population through cytotoxic killing of infected cells. Consequently, the virus population reacts through the acquisition of CTL escape mutations. Our model provides realistic virus dynamics and describes several experimental observations. We postulate that inter-clonal competition and immunodominance may be critical factors determining the sequential emergence of escapes. We show that even though the total killing induced by the CTL response can be high, escape rates against a single CTL clone are often slow and difficult to estimate from infrequent sequence measurements. Finally, our simulations show that a higher degree of immunodominance leads to more frequent escape with a reduced control of viral replication but a substantially impaired replicative capacity of the virus. This result suggests two strategies for vaccine design: Vaccines inducing a broad CTL response should decrease the viral load, whereas vaccines stimulating a narrow but dominant CTL response are likely to induce escape but may dramatically reduce the replicative capacity of the virus.     

Elicitation of high-frequency cytotoxic T-lymphocyte responses against both dominant and subdominant simian-human immunodeficiency virus epitopes by DNA vaccination of rhesus monkeys

Increasing evidence suggests that the generation of cytotoxic T-lymphocyte (CTL) responses specific for a diversity of viral epitopes will be needed for an effective human immunodeficiency virus type 1 (HIV-1) vaccine. Here, we determine the frequencies of CTL responses specific for the simian immunodeficiency virus Gag p11C and HIV-1 Env p41A epitopes in simian-human immunodeficiency virus (SHIV)-infected and vaccinated rhesus monkeys. The p11C-specific CTL response was high frequency and dominant and the p41A-specific CTL response was low frequency and subdominant in both SHIV-infected monkeys and in monkeys vaccinated with recombinant modified vaccinia virus Ankara vectors expressing these viral antigens. Interestingly, we found that plasmid DNA vaccination led to high-frequency CTL responses specific for both of these epitopes. These data demonstrate that plasmid DNA may be useful in eliciting a broad CTL response against multiple epitopes.     

EBV structural antigens, gp350 and gp85, as targets for ex vivo virus-specific CTL during acute infectious mononucleosis: potential use of gp350/gp85 CTL epitopes for vaccine design

For many years, EBV vaccine development efforts have concentrated on the use of structural Ag, gp350, and have been directed toward Ab-mediated blocking virus attachment to the target cell. There is increasing evidence to suggest that the development of neutralizing Abs in vaccinated animals does not always correlate with protection; nevertheless, it has been postulated that gp350-specific T cell-mediated immune responses may have an effector role in protection. This hypothesis has largely remained untested. In the present study, we demonstrate that CTL from acute infectious mononucleosis patients display strong ex vivo reactivity against the EBV structural Ags, gp85 and gp350. Moreover, long-term follow up studies on infectious mononucleosis-recovered individuals showed that these individuals maintain gp350- and gp85-specific memory CTL, albeit at low levels, in the peripheral blood. These results strongly suggest that CTL specific for EBV structural proteins may play an important role in the control of EBV infection during acute infection. More importantly, we also show that prior immunization of HLA A2/Kb transgenic mice with gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results have important implications for future EBV vaccine design and provides evidence, for the first time, that CTL epitopes from EBV structural proteins may be used for establishing strong antiviral immunity against EBV infection.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

Protection against establishment of retroviral persistence by vaccination with a live attenuated virus

Many human viruses not only cause acute diseases but also establish persistent infections. Such persistent viruses can cause chronic diseases or can reactivate to cause acute diseases in AIDS patients or patients receiving immunosuppressive therapies. While the prevention of persistent infections is an important consideration in the design of modern vaccines, surprisingly little is known about this aspect of protection. In the current study, we tested the feasibility of vaccine prevention of retroviral persistence by using a Friend virus model that we recently developed. In this model, persistent virus can be detected at very low levels by immunosuppressing the host to reactivate virus or by transferring persistently infected spleen cells into highly susceptible mice. Two vaccines were analyzed, a recombinant vaccinia virus vector expressing Friend virus envelope protein and a live attenuated Friend virus. Both vaccines reduced pathogenic virus loads to levels undetectable by infectious center assays. However, only the live, attenuated vaccine prevented immunosuppression-induced reactivation of persistent virus. Thus, even very low levels of persistent Friend virus posed a significant threat during immunosuppression. Our results demonstrate that vaccine protection against establishment of retroviral persistence is attainable.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

Broadly immunogenic HLA class I supertype-restricted elite CTL epitopes recognized in a diverse population infected with different HIV-1 subtypes

The genetic variations of the HIV-1 virus and its human host constitute major obstacles for obtaining potent HIV-1-specific CTL responses in individuals of diverse ethnic backgrounds infected with different HIV-1 variants. In this study, we developed and used a novel algorithm to select 184 predicted epitopes representing seven different HLA class I supertypes that together constitute a broad coverage of the different HIV-1 strains as well as the human HLA alleles. Of the tested 184 HLA class I-restricted epitopes, 114 were recognized by at least one study subject, and 45 were novel epitopes, not previously described in the HIV-1 immunology database. In addition, we identified 21 "elite" epitopes that induced CTL responses in at least 4 of the 31 patients. A majority (27 of 31) of the study population recognized one or more of these highly immunogenic epitopes. We also found a limited set of 9 epitopes that together induced HIV-1-specific CTL responses in all HIV-1-responsive patients in this study. Our results have important implications for the validation of potent CTL responses and show that the goal for a vaccine candidate in inducing broadly reactive CTL immune responses is attainable.     

Immunodominance of an antiviral cytotoxic T cell response is shaped by the kinetics of viral protein expression

Lymphocytic choriomeningitis virus (LCMV) infection induces a protective CTL response consisting of gp- and nucleoprotein (NP)-specific CTL. We find that a small load of LCMV led to immunodominance of NP-CTL, whereas a large viral load resulted in dominance of gp-CTL. This is the first study describing that immunodominance is not fixed after infection with a given pathogen, but varies with the viral load instead. We assumed higher Ag sensitivity for NP-CTL, which would explain their preferential priming at low viral load, as well as their overstimulation resulting in selective exhaustion at high viral load. The higher Ag sensitivity of NP-CTL was due to faster kinetics of NP-epitope presentation. Thus, we uncover a novel factor that impinges upon immunodominance and is related to the kinetics of virus protein expression. We propose that CTL against early viral proteins swiftly interfere with virus replication, resulting in efficient protection. If these "early" CTL fail in immediate virus control, they are activated in the face of higher viral load compared with "late" CTL and are therefore prone to be exhausted. Thus, the observed absence of early CTL in persistent infections might not be the cause, but rather the consequence of viral persistence.     

Characterization of antigen-specific immune responses induced by canarypox virus vaccines

Avipoxvirus-based vectors, such as recombinant canarypox virus ALVAC, are studied extensively as delivery vehicles for vaccines against cancer and infectious diseases. Effective use of such vaccines is expected to benefit from proper understanding of the interaction between these viral vectors and the host immune system. We performed preclinical vaccination experiments in a murine tumor model to analyze the immunogenic properties of an ALVAC-based vaccine against carcinoembryonic Ag (ALVAC-CEA), a tumor-associated autoantigen commonly overexpressed in colorectal cancers. The protective CEA-specific immunity induced by this vaccine consisted of CD4(+) T cell responses with a mixed Th1/Th2 cytokine profile that were accompanied by potent humoral responses, but not by CEA-specific CD8(+) CTL immunity. In contrast, protective immunity induced by a CEA-specific DNA vaccine (DNA-CEA) consisted of Th1 and CTL responses. Modification of the ALVAC-CEA vaccine through coinjection of DNA-CEA, admixture with CpG oligodeoxynucleotides, or supplementation with additional transgenes encoding a triad of costimulatory molecules (TRICOM) did not result in induction of CEA-specific CTL responses. Even though these results suggested that ALVAC does not elicit Ag-specific CTLs, immunization with ALVAC vaccines against other Ags efficiently induced CTL responses. Our data show that the capacity of ALVAC vaccines to elicit CTL immunity against transgene-encoded Ags critically depends on the presence of highly immunogenic CTL epitopes in these Ags. This consideration needs to be taken into account with respect to the design and evaluation of vaccination strategies that use ALVAC-based vaccine.