Efficacy of inactivated whole-virus and subunit vaccines in preventing infection and disease caused by equine infectious anemia virus.

We report here on a series of vaccine trials to evaluate the effectiveness of an inactivated equine infectious anemia virus (EIAV) whole-virus vaccine and of a subunit vaccine enriched in EIAV envelope glycoproteins. The inactivated vaccine protected 14 of 15 immunized ponies from infection after challenge with at least 10(5) 50% tissue culture-infective doses of the homologous prototype strain of EIAV. In contrast, it failed to prevent infection in any of 15 immunized ponies that were challenged with the heterologous PV strain. Levels of PV virus replication and the development of disease, however, were significantly reduced in 12 of the 15 ponies so challenged. The subunit vaccine prevented infection from homologous challenge in four of four ponies tested but failed to prevent infection in all four challenged with the PV strain. Two of the four subunit vaccinates had more severe symptoms of equine infectious anemia than nonimmunized ponies infected in parallel. Both vaccines stimulated EIAV-specific cell-mediated immunity. The in vitro lymphoproliferative response was shown to be mediated by T lymphocytes and appeared to be indistinguishable from that induced by EIAV infection. Significant differences were observed in the in vivo lymphocyte responses following challenge with the two virus strains. While peripheral blood mononuclear cells from the inactivated virus vaccinates were equally stimulated by both the prototype and PV strains, the subunit vaccinates challenged with PV exhibited lower levels of spontaneous proliferation and serine esterase activity. This diminished cellular response to PV was correlated with more severe clinical disease in the same ponies. These studies demonstrate for the first time that both an EIAV inactivated whole-virus vaccine and a viral envelope glycoprotein-based subunit vaccine can provide protection against rigorous challenge levels of homologous virus but are unable to protect against similar challenge levels of a heterologous virus. Moreover, the data demonstrate that protection can be achieved in the absence of detectable levels of virus-specific neutralizing antibody in the vaccine recipients at the time of virus challenge. While vaccine-induced virus-specific cell-mediated immune responses were detected, their role in conferring protection was not obvious. Nevertheless, protection from disease appeared to be correlated with the induction of high levels of serine esterase activity following challenge. A significant observation is that while the whole-virus vaccine was usually capable of preventing or markedly moderating disease in the PV-infected ponies, the subunit vaccine appeared to have a high potential to enhance the disease induced by PV infection.(ABSTRACT TRUNCATED AT 400 WORDS)     

Envelope Determinants of Equine Lentiviral Vaccine Protection

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy. The study identified a significant, inverse, linear correlation between vaccine efficacy and increasing divergence of the challenge virus Env gp90 protein compared to the vaccine virus gp90. The report demonstrated approximately 100% protection of immunized ponies from disease after challenge by virus with a homologous gp90 (EV0), and roughly 40% protection against challenge by virus (EV13) with a gp90 13% divergent from the vaccine strain. In the current study we examine whether the protection observed when challenging with the EV0 strain could be conferred to animals via chimeric challenge viruses between the EV0 and EV13 strains, allowing for mapping of protection to specific Env sequences. Viruses containing the EV13 proviral backbone and selected domains of the EV0 gp90 were constructed and in vitro and in vivo infectivity examined. Vaccine efficacy studies indicated that homology between the vaccine strain gp90 and the N-terminus of the challenge strain gp90 was capable of inducing immunity that resulted in significantly lower levels of post-challenge virus and significantly delayed the onset of disease. However, a homologous N-terminal region alone inserted in the EV13 backbone could not impart the 100% protection observed with the EV0 strain. Data presented here denote the complicated and potentially contradictory relationship between in vitro virulence and in vivo pathogenicity. The study highlights the importance of structural conformation for immunogens and emphasizes the need for antibody binding, not neutralizing, assays that correlate with vaccine protection.     

A live attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses

Previous evaluations of inactivated whole-virus and envelope subunit vaccines to equine infectious anemia virus (EIAV) have revealed a broad spectrum of efficacy ranging from highly type-specific protection to severe enhancement of viral replication and disease in experimentally immunized equids. Among experimental animal lentivirus vaccines, immunizations with live attenuated viral strains have proven most effective, but the vaccine efficacy has been shown to be highly dependent on the nature and severity of the vaccine virus attenuation. We describe here for the first time the characterization of an experimental attenuated proviral vaccine, EIAV(UK)deltaS2, based on inactivation of the S2 accessory gene to down regulate in vivo replication without affecting in vitro growth properties. The results of these studies demonstrated that immunization with EIAV(UK)deltaS2 elicited mature virus-specific immune responses by 6 months and that this vaccine immunity provided protection from disease and detectable infection by intravenous challenge with a reference virulent biological clone, EIAV(PV). This level of protection was observed in each of the six experimental horses challenged with the reference virulent EIAV(PV) by using a low-dose multiple-exposure protocol (three administrations of 10 median horse infectious doses [HID(50)], intravenous) designed to mimic field exposures and in all three experimentally immunized ponies challenged intravenously with a single inoculation of 3,000 HID(50). In contrast, na?ve equids subjected to the low- or high-dose challenge develop a detectable infection of challenge virus and acute disease within several weeks. Thus, these data demonstrate that the EIAV S2 gene provides an optimal site for modification to achieve the necessary balance between attenuation to suppress virulence and replication potential to sufficiently drive host immune responses to produce vaccine immunity to viral exposure.     

Immune responses and viral replication in long-term inapparent carrier ponies inoculated with equine infectious anemia virus

Persistent infection of equids by equine infectious anemia virus (EIAV) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. The goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to long-term inapparent carriage. We previously described the evolution of EIAV genomic quasispecies (C. Leroux, C. J. Issel, and R. C. Montelaro, J. Virol. 71:9627-9639, 1997) and host immune responses (S. A. Hammond, S. J. Cook, D. L. Lichtenstein, C. J. Issel, and R. C. Montelaro, J. Virol. 71:3840-3852, 1997) in four experimentally infected ponies during sequential disease episodes associated with chronic disease during the first 10 months postinfection. In the current study, we extended the studies of these experimentally infected ponies to 3 years postinfection to characterize the levels of virus replication and development of host immune responses associated with the progression from chronic disease to long-term inapparent infection. The results of these studies revealed over a 10(3)-fold difference in the steady-state levels of plasma viral RNA detected during long-term inapparent infection that correlated with the severity of chronic disease, indicating different levels of control of virus replication during long-term inapparent infections. Detailed analyses of antibody and cellular immune responses in all four ponies over the 3-year course of infection revealed a similar evolution during the first year postinfection of robust humoral and cellular immunity that then remained relatively constant during long-term inapparent infection. These observations indicate that immune parameters that have previously been correlated with EIAV vaccine protection fail to provide reliable immune correlates of control of virus replication or clinical outcome in experimental infections. Thus, these data emphasize the differences between immunity to virus exposure and immune control of an established viral infection and further emphasize the need to develop and evaluate novel immunoassays to define reliable immune correlates to vaccine and infection immunity, respectively.     

Detailed mapping of the antigenicity of the surface unit glycoprotein of equine infectious anemia virus by using synthetic peptide strategies.

We describe here a detailed analysis of the antigenic determinants of the surface unit glycoprotein (gp90) of equine infectious anemia virus (EIAV), using a comprehensive panel of synthetic peptides in enzyme-linked immunosorbent assays with immune serum from naturally and experimentally infected horses and with a panel of gp90-specific neutralizing and nonneutralizing monoclonal antibodies. The results of these studies identify immunoreactive segments throughout the conserved and variable domains of gp90 but localize immunodominant (100% reactivity) determinants to the amino and carboxyl termini of the glycoprotein molecule. Analysis of peptide reactivities with longitudinal serum samples taken from experimentally infected ponies revealed that antibody responses to conserved B-cell determinants appeared earlier and at higher titers than do antibodies specific for determinants contained in the variable domain of gp90. These observations suggest an evolution of antibody responses in EIAV-infected ponies that may correspond to the establishment of immunological control of virus replication and disease routinely observed in EIAV infections. In addition, the mapping of monoclonal antibody epitopes to peptides of 9 to 12 amino acids demonstrated that all of the neutralizing epitopes are located in the variable domain of gp90. The arrangement of neutralizing epitopes and critical structural considerations suggest that EIAV gp90 contains a principal neutralizing domain similar to the V3 loop of human immunodeficiency virus type 1. These antigenic analyses provide an important foundation for further analyzing the protective immune response generated during persistent EIAV infections and also provide potential peptide substrates for diagnostic assays and for vaccine strategies.     

Protective Efficacy of Centralized and Polyvalent Envelope Immunogens in an Attenuated Equine Lentivirus Vaccine

Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data. We previously reported on a live-attenuated (attenuated) equine infectious anemia (EIAV) virus vaccine, which provides 100% protection from disease after virulent, homologous, virus challenge. Further, protective efficacy demonstrated a significant, inverse, linear relationship between EIAV Env divergence and protection from disease when vaccinates were challenged with viral strains of increasing Env divergence from the vaccine strain Env. Here, we sought to comprehensively examine the protective efficacy of centralized immunogens in our attenuated vaccine platform. We developed, constructed, and extensively tested a consensus Env, which in a virulent proviral backbone generated a fully replication-competent pathogenic virus, and compared this consensus Env to an ancestral Env in our attenuated proviral backbone. A polyvalent attenuated vaccine was established for comparison to the centralized vaccines. Additionally, an engineered quasispecies challenge model was created for rigorous assessment of protective efficacy. Twenty-four EIAV-naïve animals were vaccinated and challenged along with six-control animals six months post-second inoculation. Pre-challenge data indicated the consensus Env was more broadly immunogenic than the Env of the other attenuated vaccines. However, challenge data demonstrated a significant increase in protective efficacy of the polyvalent vaccine. These findings reveal, for the first time, a consensus Env immunogen that generated a fully-functional, replication-competent lentivirus, which when experimentally evaluated, demonstrated broader immunogenicity that does not equate to higher protective efficacy.     

Discerning an effective balance between equine infectious anemia virus attenuation and vaccine efficacy

Among the diverse experimental vaccines evaluated in various animal lentivirus models, live attenuated vaccines have proven to be the most effective, thus providing an important model for examining critical immune correlates of protective vaccine immunity. We previously reported that an experimental live attenuated vaccine for equine infectious anemia virus (EIAV), based on mutation of the viral S2 accessory gene, elicited protection from detectable infection by virulent virus challenge (F. Li et al., J. Virol. 77:7244-7253, 2003). To better understand the critical components of EIAV vaccine efficacy, we examine here the relationship between the extent of virus attenuation, the maturation of host immune responses, and vaccine efficacy in a comparative study of three related attenuated EIAV proviral vaccine strains: the previously described EIAV(UK)DeltaS2 derived from a virulent proviral clone, EIAV(UK)DeltaS2/DU containing a second gene mutation in the virulent proviral clone, and EIAV(PR)DeltaS2 derived from a reference avirulent proviral clone. Inoculations of parallel groups of eight horses resulted in relatively low levels of viral replication (average of 10(2) to 10(3) RNA copies/ml) and a similar maturation of EIAV envelope-specific antibody responses as determined in quantitative and qualitative serological assays. However, experimental challenge of the experimentally immunized horses by our standard virulent EIAV(PV) strain by using a low-dose multiple exposure protocol (three inoculations with 10 median horse infective doses, administered intravenously) revealed a marked difference in the protective efficacy of the various attenuated proviral vaccine strains that was evidently associated with the extent of vaccine virus attenuation, time of viral challenge, and the apparent maturation of virus-specific immunity.     

EIAV env基因在痘苗病毒中的表达及其免疫效果的观察

将马传贫驴白细胞弱毒疫苗及其亲本株env基因克隆到痘苗病毒表达载体pSC65的pE／L启动子下游,通过同源重组插入到痘苗病毒天坛株基因组TK区,经蓝白斑筛选获得重组痘苗病毒rvv—DINenv和rvv—LNenv,Westem blot检测目的蛋白的表达,结果表明重组痘苗病毒能够有效表达完整的EIAV Env蛋白,其肌肉接种免疫小鼠后,表达的目的蛋白具有良好的免疫原性,能够诱导机体产生有效的体液和细胞免疫应答,其中以细胞免疫效果更为显著,CTL特异性裂解最高可达28％。本研究为EIAV基因工程疫苗的开发研制奠定了基础。     

Maturation of the cellular and humoral immune responses to persistent infection in horses by equine infectious anemia virus is a complex and lengthy process.

Equine infectious anemia virus (EIAV) provides a natural model system by which immunological control of lentivirus infections may be studied. To date, no detailed study addressing in parallel both the humoral and cellular immune responses induced in horses upon infection by EIAV has been conducted. Therefore, we initiated the first comprehensive characterization of the cellular and humoral immune responses during clinical progression from chronic disease to inapparent stages of EIAV infection. Using new analyses of antibody avidity and antibody epitope conformation dependence that had not been previously employed in this system, we observed that the humoral immune response to EIAV required a 6- to 8-month period in which to fully mature. During this time frame, EIAV-specific antibody evolved gradually from a population characterized by low-avidity, nonneutralizing, and predominantly linear epitope specificity to an antibody population with an avidity of moderate to high levels, neutralizing activity, and predominantly conformational epitope specificity. Analyses of the cell-mediated immune response to EIAV revealed CD4+ and CD8+ major histocompatibility complex-restricted, EIAV-specific cytotoxic T-lymphocyte (CTL) activity apparent within 3 to 4 weeks postinfection, temporally correlating with the resolution of the primary viremia. After resolution of the initial viremia, EIAV-specific CTL activity differed greatly among the experimentally infected ponies, with some animals having readily detectable CTL activity while others had little measurable CTL activity. Thus, in contrast to the initial viremia, it appeared that no single immune parameter correlated with the resolution of further viremic episodes. Instead, immune control of EIAV infection during the clinically inapparent stage of infection appears to rely on a complex combination of immune system mechanisms to suppress viral replication that effectively functions only after the immune system has evolved to a fully mature state 6 to 8 months postinfection. These findings strongly imply the necessity for candidate EIAV and other lentivirus vaccines to achieve this immune system maturation for efficacious immunological control of lentivirus challenge.     

Heterologous envelope immunogens contribute to AIDS vaccine protection in rhesus monkeys

Because a strategy to elicit broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies has not yet been found, the role of an Env immunogen in HIV-1 vaccine candidates remains undefined. We sought to determine whether an HIV-1 Env immunogen genetically disparate from the Env of the challenge virus can contribute to protective immunity. We vaccinated Indian-origin rhesus monkeys with Gag-Pol-Nef immunogens, alone or in combination with Env immunogens that were either matched or mismatched with the challenge virus. These animals were then challenged with a pathogenic simian-human immunodeficiency virus. The vaccine regimen included a plasmid DNA prime and replication-defective adenoviral vector boost. Vaccine regimens that included the matched or mismatched Env immunogens conferred better protection against CD4(+) T-lymphocyte loss than that seen with comparable regimens that did not include Env immunogens. This increment in protective immunity was associated with anamnestic Env-specific cellular immunity that developed in the early days following viral challenge. These data suggest that T-lymphocyte immunity to Env can broaden the protective cellular immune response to HIV despite significant sequence diversity of the strains of the Env immunogens and can contribute to immune protection in this AIDS vaccine model.     

Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (delta5G). In delta5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and delta5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in delta5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in delta5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.     

Role of Humoral versus Cellular Responses Induced by a Protective Dengue Vaccine Candidate

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.     

Incorporation of membrane-anchored flagellin into influenza virus-like particles enhances the breadth of immune responses

We have designed a membrane-anchored form of the Toll-like receptor 5 ligand flagellin, the major proinflammatory determinant of enteropathogenic Salmonella, which was found to be glycosylated and expressed on cell surfaces. A chimeric influenza virus-like particle (cVLP) vaccine candidate containing A/PR8/34 (H(1)N(1)) hemagglutinin (HA), matrix protein (M1), and the modified flagellin as a molecular adjuvant was produced. The immunogenicity, including the serum antibody levels and cellular immune responses, and the protective efficacy against homologous and heterologous live virus challenge of the resulting VLPs were tested after intramuscular administration in a mouse model. The results demonstrated that flagellin-containing VLPs elicited higher specific immunoglobulin G (IgG) responses than standard HA and M1 VLPs, indicating the adjuvant effect of flagellin. Enhanced IgG2a and IgG2b but not IgG1 responses were observed with flagellin-containing VLPs, illuminating the activation of Th1 class immunity. The adjuvant effects of flagellin were also reflected by enhanced specific cellular responses revealed by the secretion of cytokines by freshly isolated splenocyte cultures when stimulated with pools of major histocompatibility complex class I or II peptides. When immunized mice were challenged with homologous live PR8 virus, complete protection was observed for both the standard and cVLP groups. However, when a heterosubtypic A/Philippines (H(3)N(2)) virus was used for challenge, all of the standard VLP group lost at least 25% of body weight, reaching the experimental endpoint. In contrast, for the cVLP group, 67% of mice survived the challenge infection. These results reveal that cVLPs designed by incorporating flagellin as a membrane-anchored adjuvant induce enhanced cross-protective heterosubtypic immune responses. They also indicate that such cVLP vaccines are a promising new approach for protection against pandemic influenza viruses.     

Modifications of the human immunodeficiency virus envelope glycoprotein enhance immunogenicity for genetic immunization

In this study, we have investigated the effect of specific mutations in human immunodeficiency virus type 1 (HIV-1) envelope (Env) on antibody production in an effort to improve humoral immune responses to this glycoprotein by DNA vaccination. Mice were injected with plasmid expression vectors encoding HIV Env with modifications in regions that might affect this response. Elimination of conserved glycosylation sites did not substantially enhance humoral or cytotoxic-T-lymphocyte (CTL) immunity. In contrast, a modified gp140 with different COOH-terminal mutations intended to mimic a fusion intermediate and stabilize trimer formation enhanced humoral immunity without reducing the efficacy of the CTL response. This mutant, with deletions in the cleavage site, fusogenic domain, and spacing of heptad repeats 1 and 2, retained native antigenic conformational determinants as defined by binding to known monoclonal antibodies or CD4, oligomer formation, and virus neutralization in vitro. Importantly, this modified Env, gp140 Delta CFI, stimulated the antibody response to native gp160 while it retained its ability to induce a CTL response, a desirable feature for an AIDS vaccine.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

Analysis of the human env-specific cytotoxic T-lymphocyte (CTL) response in natural human immunodeficiency virus type 1 infection: low prevalence of broadly cross-reactive env-specific CTL.

Major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) are part of the cellular immune response to persistent virus infections. Candidate vaccines against human immunodeficiency virus type 1 (HIV-1) should elicit broad cross-reactive immunity to confer protection against different strains of HIV-1. As it is likely that candidate vaccines will include the envelope gene product Env, we determined the proportion of CTL clones which recognized variable and conserved determinants in three env variants during natural infection. Limiting dilution analysis was used to characterize numerous short-term CTL clones derived from peripheral blood of HIV-1-infected subjects, using split-well analysis to assay cytotoxicity against target cells expressing gp160env of HIV-1 strains IIIB, MN, and RF. In 9 of 12 HIV-1-infected subjects, at the clonal level most env-specific CTL recognized determinant(s) within one env variant but not in the other variants. In some subjects, CTL recognized multiple nonconserved determinants in different variants. The pattern of recognition of different env variants was relatively stable over time. In most of the patients studied, the proportion of CTL which showed cross-recognition of conserved determinants shared among the three strains was low. Two novel CTL epitopes within gp41 were identified by using 15-mer peptides of the HIV-SF2 sequence. When specific peptide was used to stimulate CTL precursors in vitro, the frequency of peptide-specific CTL precursors was very high, but the CTL elicited by this stimulation were highly strain specific. We conclude that the use of a single HIV env variant to detect CTL activity can underestimate the magnitude and complexity of the env-specific CTL response. The low prevalence of CTL clones which show cross-recognition of conserved determinants may have implications for immunization strategies based solely on env; to elicit broadly cross-reactive CTL other, more conserved viral antigens are likely to be needed in addition to env. Because of its capacity to distinguish CTL responses against different virus strains, limiting dilution analysis is particularly appropriate to quantitate the immune responses generated by candidate env-based vaccines.     

Gag Protein Epitopes Recognized by ELA-A-Restricted Cytotoxic T Lymphocytes from Horses with Long-Term Equine Infectious Anemia Virus Infection

Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.Equine infectious anemia virus (EIAV) belongs to the Lentivirus genus, which includes human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and several other animal viruses. EIAV causes disease in horses which is characterized by recurrent febrile episodes associated with viremia, anemia, and thrombocytopenia (10). Most infected horses are able to eventually control the disease and become lifelong EIAV carriers (9). The ability of horses to restrict EIAV replication to very low levels and to remain free of clinical disease provides an opportunity to determine the immunologic mechanisms involved in this lentivirus control.Immune responses are required for the termination of the acute viremia during EIAV infection since foals with severe combined immunodeficiency cannot control the initial viremia following EIAV infection, in contrast to normal foals (41). Results suggesting that immune responses are involved in the control of EIAV in carrier horses include the observation that corticosteroid- and cyclophosphamide-treated carrier horses have recurrent viremia and disease (24). Neutralizing antibody can be an important component of the protective immune response against lentiviral infections (12). Type-specific neutralizing antibody appears following the episodes of plasma viremia in EIAV-infected horses (25); however, there is evidence suggesting that the presence of the neutralizing antibody does not necessarily relate to the occurrence and control of viremic episodes (8, 25). Detectable neutralizing antibodies to the variant isolated during a disease episode can appear after the episode is controlled (8). Neutralizing antibody-escape variants are isolated from EIAV carrier horses as early as 5 days after corticosteroid treatment, when the antibody levels have not significantly changed (24). Further, the viremic episode induced by corticosteroid treatment can be terminated before the appearance of neutralizing antibody to the variant causing viremia (24). Other evidence implicating immune responses other than neutralizing antibody in EIAV control includes the following: (i) EIAV carrier horses can resist challenge with a heterologous strain in the absence of detectable neutralizing antibody to the challenge virus (23), and (ii) some horses immunized with an inactivated virus vaccine resist homologous strain challenge without detectable levels of neutralizing antibody but with virus-specific cell-mediated immune responses (17).Accumulating evidence suggests that major histocompatibility complex (MHC) class I-restricted virus-specific cytotoxic T lymphocytes (CTL) may play an important role in the immune control of diseases caused by HIV-1 and SIV infection (5, 26, 51). CTL appear to be involved in both the clearance of the primary viremia in HIV-1 infection (26) and the prevention of disease progression to AIDS (42). In EIAV infection, the appearance of activated CD8⁺ CTL (effectors) correlated with the control of the initial viremic episodes (33). Although the CTL effectors decline to low levels when plasma viremias become undetectable, a high frequency of memory CTL (CTLm) has been detected in some carrier horses (34), and these CTLm recognize either EIAV Env or Gag/Pr proteins or both (15, 34). Both CD8⁺ and CD4⁺ CTL activities have been detected in some EIAV-infected horses (15), but their roles in disease control are not known.The epitopes recognized by CD8⁺ CTL are usually peptides of 8 to 11 amino acids (aa) presented by MHC class I molecules on the target cell surface. Identifying the CTL epitopes and the MHC class I molecules that restrict responses is necessary in order to determine how CTL are involved in the control of disease and to stimulate CTL by vaccination. However, the occurrence of escape mutants which are no longer recognized by CTL is one of the major difficulties for inducing effective CTL responses against different variants (6). Gag protein epitopes recognized by CTL may be of importance because Gag proteins are relatively conserved among EIAV strains (21, 32, 40, 48). In this study, at least 12 peptides with CTL epitopes were recognized by stimulated peripheral blood mononuclear cells (PBMC) from six long-term EIAV-infected horses with different ELA-A alleles. These peptides were identified by using retroviral vectors expressing individual Gag proteins and synthetic overlapping peptides from recognized proteins. We identified two nonamer peptides, one apparently restricted by ELA-A5.1, and another by ELA-A9, molecules.     

Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination

Since cytotoxic T lymphocytes (CTLs) are critical for controlling human immunodeficiency virus type 1 (HIV-1) replication in infected individuals, candidate HIV-1 vaccines should elicit virus-specific CTL responses. In this report, we study the immune responses elicited in rhesus monkeys by a recombinant poxvirus vaccine and the degree of protection afforded against a pathogenic simian-human immunodeficiency virus SHIV-89.6P challenge. Immunization with recombinant modified vaccinia virus Ankara (MVA) vectors expressing SIVmac239 gag-pol and HIV-1 89.6 env elicited potent Gag-specific CTL responses but no detectable SHIV-specific neutralizing antibody (NAb) responses. Following intravenous SHIV-89.6P challenge, sham-vaccinated monkeys developed low-frequency CTL responses, low-titer NAb responses, rapid loss of CD4+ T lymphocytes, high-setpoint viral RNA levels, and significant clinical disease progression and death in half of the animals by day 168 postchallenge. In contrast, the recombinant MVA-vaccinated monkeys demonstrated high-frequency secondary CTL responses, high-titer secondary SHIV-89.6-specific NAb responses, rapid emergence of SHIV-89.6P-specific NAb responses, partial preservation of CD4+ T lymphocytes, reduced setpoint viral RNA levels, and no evidence of clinical disease or mortality by day 168 postchallenge. There was a statistically significant correlation between levels of vaccine-elicited CTL responses prior to challenge and the control of viremia following challenge. These results demonstrate that immune responses elicited by live recombinant vectors, although unable to provide sterilizing immunity, can control viremia and prevent disease progression following a highly pathogenic AIDS virus challenge.     

Codelivery of the chemokine CCL3 by an adenovirus-based vaccine improves protection from retrovirus infection

Processing and presentation of vaccine antigens by professional antigen-presenting cells (APCs) is of great importance for the efficient induction of protective immunity. We analyzed whether the efficacy of an adenovirus-based retroviral vaccine can be enhanced by coadministration of adenovirus-encoded chemokines that attract and stimulate APCs. In the Friend retrovirus (FV) mouse model we coexpressed CCL3, CCL20, CCL21, or CXCL14 from adenoviral vectors, together with FV Gag and Env antigens, and then analyzed immune responses and protection from pathogenic FV infection. Although most tested chemokines did not improve protection against FV challenge, mice that received adenoviral vectors encoding CCL3 together with FV antigens showed significantly better control over viral loads and FV-induced disease than mice immunized with the viral antigens only. Improved protection correlated with enhanced virus-specific CD4+ T cell responses and higher neutralizing antibody titers. To apply these results to an HIV vaccine, mice were immunized with adenoviral vectors encoding the HIV antigens Env and Gag-Pol and coadministered vectors encoding CCL3. Again, this combination vaccine induced higher virus-specific antibody titers and CD4+ T cell responses than did the HIV antigens alone. These results indicate that coexpression of the chemokine CCL3 by adenovirus-based vectors may be a promising tool to improve antiretroviral vaccination strategies.     

Influence of Mismatch of Env Sequences on Vaccine Protection by Live Attenuated Simian Immunodeficiency Virus

Vaccine/challenge experiments that utilize live attenuated strains of simian immunodeficiency virus (SIV) in monkeys may be useful for elucidating what is needed from a vaccine in order to achieve protective immunity. Derivatives of SIVmac239 and SIVmac239Δnef were constructed in which env sequences were replaced with those of the heterologous strain E543; these were then used in vaccine/challenge experiments. When challenge occurred at 22 weeks, 10 of 12 monkeys exhibited apparent sterilizing immunity despite a mismatch of Env sequences, compared to 12 of 12 monkeys with apparent sterilizing immunity when challenge virus was matched in its Env sequence. However, when challenge occurred at 6 weeks, 6 of 6 SIV239Δnef-immunized monkeys became superinfected by challenge virus mismatched in its Env sequence (SIV239/EnvE543). These results contrast markedly not only with the results of the week 22 challenge but also with the sterilizing immunity observed in 5 of 5 SIV239Δnef-immunized rhesus monkeys challenged at 5 weeks with SIV239, i.e., with no mismatch of Env sequences. We conclude from these studies that a mismatch of Env sequences in the challenge virus can have a dramatic effect on the extent of apparent sterilizing immunity when challenge occurs relatively early, 5 to 6 weeks after the nef-deleted SIV administration. However, by 22 weeks, mismatch of Env sequences has little or no influence on the degree of protection against challenge virus. Our findings suggest that anti-Env immune responses are a key component of the protective immunity elicited by live attenuated, nef-deleted SIV.