Antiviral activity of human immunodeficiency virus type 1 Gag-specific cytotoxic T lymphocyte targeting is not necessarily intrinsically superior to envelope targeting

Across several cohorts, human immunodeficiency virus type 1 (HIV-1) Gag- and Env-specific CD8(+) T lymphocyte (CTL) responses have demonstrated inverse and positive correlations, respectively, to viremia. The mechanism has been proposed to be superior antiviral activity of Gag-specific CTLs in general. Addressing this hypothesis, we created two HIV-1 constructs with an epitope translocated from Gag (SLYNTVATL, SL9) to Env, thereby switching the protein source of the epitope. A virus expressing SL9 in Env was similar to the original virus in susceptibility to SL9-specific CTLS. This finding suggests that Env targeting is not intrinsically inferior to Gag targeting for CTL antiviral activity.     

Gag-specific CD8+ T lymphocytes recognize infected cells before AIDS-virus integration and viral protein expression

CD8(+) T cells are a key focus of vaccine development efforts for HIV. However, there is no clear consensus as to which of the nine HIV proteins should be used for vaccination. The early proteins Tat, Rev, and Nef may be better CD8(+) T cell targets than the late-expressed structural proteins Gag, Pol, and Env. In this study, we show that Gag-specific CD8(+) T cells recognize infected CD4(+) T lymphocytes as early as 2 h postinfection, before proviral DNA integration, viral protein synthesis, and Nef-mediated MHC class I down-regulation. Additionally, the number of Gag epitopes recognized by CD8(+) T cells was significantly associated with lower viremia (p = 0.0017) in SIV-infected rhesus macaques. These results suggest that HIV vaccines should focus CD8(+) T cell responses on Gag.     

The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens

Attenuated poxvirus vectors expressing human immunodeficiency virus type 1 (HIV-1) antigens are considered promising HIV/AIDS vaccine candidates. Here, we describe the nature of T cell immune responses induced in healthy volunteers participating in a phase I clinical trial in Spain after intramuscular administration of three doses of the recombinant MVA-B-expressing monomeric gp120 and the fused Gag-Pol-Nef (GPN) polyprotein of clade B. The majority (92.3%) of the volunteers immunized had a positive specific T cell response at any time postvaccination as detected by gamma interferon (IFN-γ) intracellular cytokine staining (ICS) assay. The CD4(+) T cell responses were predominantly Env directed, whereas the CD8(+) T cell responses were similarly distributed against Env, Gag, and GPN. The proportion of responders after two doses of MVA-B was similar to that obtained after the third dose of MVA-B vaccination, and the responses were sustained (84.6% at week 48). Vaccine-induced CD8(+) T cells to HIV-1 antigens after 1 year were polyfunctional and distributed mainly within the effector memory (TEM) and terminally differentiated effector memory (TEMRA) T cell populations. Antivector T cell responses were mostly induced by CD8(+) T cells, highly polyfunctional, and of TEMRA phenotype. These findings demonstrate that the poxvirus MVA-B vaccine candidate given alone is highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4 and CD8 T cell responses to HIV-1 antigens, with preference for TEM. Thus, on the basis of the immune profile of MVA-B in humans, this immunogen can be considered a promising HIV/AIDS vaccine candidate.     

Evaluation of Novel Human Immunodeficiency Virus Type 1 Gag DNA Vaccines for Protein Expression in Mammalian Cells and Induction of Immune Responses

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) are an important parameter of host defenses that limit viral replication after infection. Induction of effective CTL against conserved viral proteins such as Gag may be essential to the development of a safe and effective HIV type 1 (HIV-1) vaccine. DNA vaccination represents a novel strategy for inducing potent CD8(+) CTL responses in vivo. However, expression of HIV-1 structural proteins by DNA vectors has been hampered by a stringent requirement for coexpression with other viral components, such as Rev and RRE. Furthermore, even with Rev and RRE present, the level of expression of HIV-1 Gag, Pol, or Env is very low in murine cells. These problems have limited our ability to address the key issue of how to generate effective CTL responses to Gag in a mouse model. To overcome this problem, we compared several novel DNA expression vectors for HIV-1 Gag protein expression in primate and mouse cells and for generating immune responses in mice after DNA vaccination. A DNA vector containing wild type HIV-1 gag coding sequences did not induce detectable Gag expression in any of the cells tested. Attempts to increase nuclear export of Gag expression RNA by adding the constitutive transport element yielded only a moderate increase in Gag expression in monkey-derived COS cells and an even lower increase in Gag expression in HeLa cells or several mouse cell lines. In contrast, silent-site mutations in the HIV-1 gag coding sequences significantly increased Gag expression levels in all cells tested. Furthermore, this construct induced both Gag-specific antibody and CTL responses in mice after DNA vaccination. Using this construct, we achieved stable expression of HIV-1 Gag in the mouse cell line p815, which can now be used as a target cell for measuring HIV-1 Gag-specific CTL responses in immunized mice. The DNA vectors described in this study should make it possible to systematically evaluate the approaches for maximizing the induction of CTL responses against HIV-1 Gag in mouse and other animal systems.     

Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine

Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4(+) cells. Interestingly, most of the CD4(+) cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4(+) cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4(+) cells.     

Gag-Specific Cellular Immunity Determines In Vitro Viral Inhibition and In Vivo Virologic Control following Simian Immunodeficiency Virus Challenges of Vaccinated Rhesus Monkeys

A comprehensive vaccine for human immunodeficiency virus type 1 (HIV-1) would block HIV-1 acquisition as well as durably control viral replication in breakthrough infections. Recent studies have demonstrated that Env is required for a vaccine to protect against acquisition of simian immunodeficiency virus (SIV) in vaccinated rhesus monkeys, but the antigen requirements for virologic control remain unclear. Here, we investigate whether CD8(+) T lymphocytes from vaccinated rhesus monkeys mediate viral inhibition in vitro and whether these responses predict virologic control following SIV challenge. We observed that CD8(+) lymphocytes from 23 vaccinated rhesus monkeys inhibited replication of SIV in vitro. Moreover, the magnitude of inhibition prior to challenge was inversely correlated with set point SIV plasma viral loads after challenge. In addition, CD8 cell-mediated viral inhibition in vaccinated rhesus monkeys correlated significantly with Gag-specific, but not Pol- or Env-specific, CD4(+) and CD8(+) T lymphocyte responses. These findings demonstrate that in vitro viral inhibition following vaccination largely reflects Gag-specific cellular immune responses and correlates with in vivo virologic control following infection. These data suggest the importance of including Gag in an HIV-1 vaccine in which virologic control is desired.     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

Utility of human immunodeficiency virus type 1 envelope as a T-cell immunogen

Human immunodeficiency virus (HIV)-specific CD8 T lymphocytes are important for the control of viremia, but the relative utility of responses to the various HIV proteins is controversial. Immune responses that force escape mutations that exact a significant fitness cost from the mutating virus would help slow progression to AIDS. The HIV envelope (Env) protein is subject to both humoral and cellular immune responses, suggesting that multiple rounds of mutation are needed to facilitate viral escape. The Gag protein, however, has recently been shown to elicit a more effective CD8 T-cell immune response in humans. We studied 30 pigtail macaques for their CD8 T-lymphocyte responses to HIV-1 Env and simian immunodeficiency virus (SIV) Gag following prime/boost vaccination and intrarectal challenge with simian-human immunodeficiency virus SHIV_mn229. Eight CD8 Env-specific T-cell epitopes were identified and mapped in 10 animals. Animals that generated Env-specific CD8 T-cell responses had equivalent viral loads and only a modest advantage in retention of peripheral CD4 T lymphocytes compared to those animals without responses to Env. This contrasts with animals that generated CD8 T-cell responses to SIV Gag in the same trial, demonstrating superior control of viral load and a larger advantage in retention of peripheral CD4 T cells than Gag nonresponders. Mutational escape was common in Env but, in contrast to mutations in Gag, did not result in the rapid emergence of dominant escape motifs, suggesting modest selective pressure from Env-specific T cells. These results suggest that Env may have limited utility as a CD8 T-cell immunogen.     

HIV-specific CD4 T cell responses to different viral proteins have discordant associations with viral load and clinical outcome

A successful prophylactic vaccine is characterized by long-lived immunity, which is critically dependent on CD4 T cell-mediated helper signals. Indeed, most licensed vaccines induce antigen-specific CD4 T cell responses, in addition to high-affinity antibodies. However, despite the important role of CD4 T cells in vaccine design and natural infection, few studies have characterized HIV-specific CD4 T cells due to their preferential susceptibility to HIV infection. To establish at the population level the impact of HIV-specific CD4 T cells on viral control and define the specificity of HIV-specific CD4 T cell peptide targeting, we conducted a comprehensive analysis of these responses to the entire HIV proteome in 93 subjects at different stages of HIV infection. We show that HIV-specific CD4 T cell responses were detectable in 92% of individuals and that the breadth of these responses showed a significant inverse correlation with the viral load (P = 0.009, R = -0.31). In particular, CD4 T cell responses targeting Gag were robustly associated with lower levels of viremia (P = 0.0002, R = -0.45). Importantly, differences in the immunodominance profile of HIV-specific CD4 T cell responses distinguished HIV controllers from progressors. Furthermore, Gag/Env ratios were a potent marker of viral control, with a high frequency and magnitude of Gag responses and low proportion of Env responses associated with effective immune control. At the epitope level, targeting of three distinct Gag peptides was linked to spontaneous HIV control (P = 0.60 to 0.85). Inclusion of these immunogenic proteins and peptides in future HIV vaccines may act as a critical cornerstone for enhancing protective T cell responses.     

Enhanced potency of plasmid DNA microparticle human immunodeficiency virus vaccines in rhesus macaques by using a priming-boosting regimen with recombinant proteins

DNA vaccines have been used widely in experimental primate models of human immunodeficiency virus (HIV), but their effectiveness has been limited. In this study, we evaluated three technologies for increasing the potency of DNA vaccines in rhesus macaques. These included DNA encoding Sindbis virus RNA replicons (pSINCP), cationic poly(lactide-co-glycolide) (PLG) microparticles for DNA delivery, and recombinant protein boosting. The DNA-based pSINCP replicon vaccines encoding HIV Gag and Env were approximately equal in potency to human cytomegalovirus (CMV) promoter-driven conventional DNA vaccines (pCMV). The PLG microparticle DNA delivery system was particularly effective at enhancing antibody responses induced by both pCMV and pSINCP vaccines and had less effect on T cells. Recombinant Gag and Env protein boosting elicited rapid and strong recall responses, in some cases to levels exceeding those seen after DNA or DNA/PLG priming. Of note, Env protein boosting induced serum-neutralizing antibodies and increased frequencies of gamma interferon-producing CD4 T cells severalfold. Thus, PLG microparticles are an effective means of delivering DNA vaccines in nonhuman primates, as demonstrated for two different types of DNA vaccines encoding two different antigens, and are compatible for use with DNA prime-protein boost regimens.     

Multifunctional T-cell characteristics induced by a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine regimen given to healthy adults are dependent on the route and dose of administration

A phase I clinical vaccine study of a human immunodeficiency virus type 1 (HIV-1) vaccine regimen comprising a DNA prime formulation (5-valent env and monovalent gag) followed by a 5-valent Env protein boost for seronegative adults was previously shown to induce HIV-1-specific T cells and anti-Env antibodies capable of neutralizing cross-clade viral isolates. In light of these initial findings, we sought to more fully characterize the HIV-1-specific T cells by using polychromatic flow cytometry. Three groups of participants were vaccinated three times with 1.2 mg of DNA administered intradermally (i.d.; group A), 1.2 mg of DNA administered intramuscularly (i.m.; group B), or 7.2 mg of DNA administered i.m. (high-dose group C) each time. Each group subsequently received one or two doses of 0.375 mg each of the gp120 protein boost vaccine (i.m.). Env-specific CD4 T-cell responses were seen in the majority of participants; however, the kinetics of responses differed depending on the route of DNA administration. The high i.m. dose induced the responses of the greatest magnitude after the DNA vaccinations, while the i.d. group exhibited the responses of the least magnitude. Nevertheless, after the second protein boost, the magnitude of CD4 T-cell responses in the i.d. group was indistinguishable from those in the other two groups. After the DNA vaccinations and the first protein boost, a greater number of polyfunctional Env-specific CD4 T cells (those with > or = 2 functions) were seen in the high-dose group than in the other groups. Gag-specific CD4 T cells and Env-specific CD8 T cells were seen only in the high-dose group. These findings demonstrate that the route and dose of DNA vaccines significantly impact the quality of immune responses, yielding important information for future vaccine design.     

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.     

Mycobacterium tuberculosis Infection Interferes with HIV Vaccination in Mice

Tuberculosis (TB) has emerged as the most prominent bacterial disease found in human immunodeficiency virus (HIV)-positive individuals worldwide. Due to high prevalence of asymptomatic Mycobacterium tuberculosis (Mtb) infections, the future HIV vaccine in areas highly endemic for TB will often be administrated to individuals with an ongoing Mtb infection. The impact of concurrent Mtb infection on the immunogenicity of a HIV vaccine candidate, MultiHIV DNA/protein, was investigated in mice. We found that, depending on the vaccination route, mice infected with Mtb before the administration of the HIV vaccine showed impairment in both the magnitude and the quality of antibody and T cell responses to the vaccine components p24Gag and gp160Env. Mice infected with Mtb prior to intranasal HIV vaccination exhibited reduced p24Gag-specific serum IgG and IgA, and suppressed gp160Env-specific serum IgG as compared to respective titers in uninfected HIV-vaccinated controls. Importantly, in Mtb-infected mice that were HIV-vaccinated by the intramuscular route the virus neutralizing activity in serum was significantly decreased, relative to uninfected counterparts. In addition mice concurrently infected with Mtb had fewer p24Gag-specific IFN-γ-expressing T cells and multifunctional T cells in their spleens. These results suggest that Mtb infection might interfere with the outcome of prospective HIV vaccination in humans.     

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.     

GagCM9-specific CD8+ T cells expressing limited public TCR clonotypes do not suppress SIV replication in vivo

Several lines of evidence suggest that HIV/SIV-specific CD8(+) T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag(45-269)) that were subsequently infected with SIVsmE660. These seven Mamu-A*01(+) animals developed CD8(+) T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8(+) T cells could not control virus replication in vivo. GagCM9-specific CD8(+) T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8(+) T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20-250 GagCM9-specific CD8(+) T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8(+) T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8(+) T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8(+) T cell population elicited by vaccination and infection.     

Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus

Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.     

Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys

The development of a human immunodeficiency virus type 1 (HIV-1) vaccine that elicits potent cellular and humoral immune responses recognizing divergent strains of HIV-1 will be critical for combating the global AIDS epidemic. The present studies were initiated to examine the magnitude and breadth of envelope (Env)-specific T-lymphocyte and antibody responses generated by vaccines containing either a single or multiple genetically distant HIV-1 Env immunogens. Rhesus monkeys were immunized with DNA prime-recombinant adenovirus boost vaccines encoding a Gag-Pol-Nef polyprotein in combination with either a single Env or a mixture of clade-A, clade-B, and clade-C Envs. Monkeys receiving the multiclade Env immunization developed robust immune responses to all vaccine antigens and, importantly, a greater breadth of Env recognition than monkeys immunized with vaccines including a single Env immunogen. All groups of vaccinated monkeys demonstrated equivalent immune protection following challenge with the pathogenic simian-human immunodeficiency virus 89.6P. These data suggest that a multicomponent vaccine encoding Env proteins from multiple clades of HIV-1 can generate broad Env-specific T-lymphocyte and antibody responses without antigenic interference. This study demonstrates that it is possible to generate protective immune responses by vaccination with genetically diverse isolates of HIV-1.     

Immunogenicity of DNA and recombinant Sendai virus vaccines expressing the HIV-1 gag gene

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens. Foundation item: National 863 project (2003AA219070)     

Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env(121-129) epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.