Novel Mucosal DNA-MVA HIV Vaccination in Which DNA-IL-12 Plus Cholera Toxin B Subunit (CTB) Cooperates to Enhance Cellular Systemic and Mucosal Genital Tract Immunity

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after their mucosal administration in DNA prime/MVA boost intranasal regimes, defining the cooperation of both adjuvants to enhance immune responses against the HIV-1 Env antigen. Our results demonstrated that nasal mucosal DNA/MVA immunization schemes can be effectively improved by the co-delivery of DNA-IL-12 plus CTB inducing elevated HIV-specific CD8 responses in spleen and more importantly in genital tract and genito-rectal draining lymph nodes. Remarkably, these CTL responses were of superior quality showing higher avidity, polyfunctionality and a broader cytokine profile. After IL-12+CTB co-delivery, the cellular responses induced showed an enhanced breadth recognizing with higher efficiency Env peptides from different subtypes. Even more, an in vivo CTL cytolytic assay demonstrated the higher specific CD8 T-cell performance after the IL-12+CTB immunization showing in an indirect manner its potential protective capacity. Improvements observed were maintained during the memory phase where we found higher proportions of specific central memory and T memory stem-like cells T-cell subpopulations. Together, our data show that DNA-IL-12 plus CTB can be effectively employed acting as mucosal adjuvants during DNA prime/MVA boost intranasal vaccinations, enhancing magnitude and quality of HIV-specific systemic and mucosal immune responses.     

Improving the MVA vaccine potential by deleting the viral gene coding for the IL-18 binding protein

Background

Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L).

Methodology/Principal Findings

BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8⁺ and CD4⁺ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8⁺ T-cells (CD107a/b⁺) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA''s CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens.

Conclusions/Significance

This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.     

Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV-specific CTL

We examined the interplay between cytokines and adjuvants to optimize the induction of CTL by a mucosal HIV peptide vaccine. We show synergy between IL-12 and GM-CSF when administered together with the HIV peptide PCLUS3-18IIIB and cholera toxin (CT) in the induction of CTL activity and protection against mucosal viral transmission. Further, we examine the efficacy of mutant Escherichia coli labile toxin, LT(R192G), as a less toxic adjuvant than CT. LT(R192G) was as effective as or more effective than CT at inducing a mucosal CTL response. Moreover, LT(R192G) was as effective without IL-12 as CT was when combined with IL-12, and the response elicited by LT(R192G) with the vaccine was not further enhanced by the addition of IL-12. GM-CSF synergized with LT(R192G) without exogenous IL-12. Therefore, LT(R192G) may induce a more favorable cytokine response by not inhibiting IL-12 production. In particular, less IL-4 is made after LT(R192G) than CT immunization, and the response is less susceptible to anti-IL-12 inhibition. Thus, the choice of mucosal adjuvant affects the cytokine environment, and the mucosal response and protection can be enhanced by manipulating the cytokine environment with synergistic cytokine combinations incorporated in the vaccine.     

Effects of Antigen and Genetic Adjuvants on Immune Responses to Human Immunodeficiency Virus DNA Vaccines in Mice

The effects of genetic adjuvants on humoral and cell-mediated immunity to two human immunodeficiency virus antigens, Env and Nef, have been examined in mice. Despite similar levels of gene expression and the same gene delivery vector, the immune responses to these two gene products differed following DNA immunization. Intramuscular immunization with a Nef expression vector plasmid generated a humoral response and antigen-specific gamma interferon (IFN-gamma) production but little cytotoxic-T-lymphocyte (CTL) immunity. In contrast, immunization with an Env vector stimulated CTL activity but did not induce a high-titer antibody response. The ability to modify these antigen-specific immune responses was investigated by coinjection of DNA plasmids encoding cytokine and/or hematopoietic growth factors, interleukin-2 (IL-2), IL-12, IL-15, Flt3 ligand (FL), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Coadministration of these genes largely altered the immune responses quantitatively but not qualitatively. IL-12 induced the greatest increase in IFN-gamma and immunoglobulin G responses to Nef, and GM-CSF induced the strongest IFN-gamma and CTL responses to Env. A dual approach of expanding innate immunity by administering the FL gene, together with a cytokine that enhances adaptive immune responses, IL-2, IL-12, or IL-15, generated the most potent immune response at the lowest doses of Nef antigen. These findings suggest that intrinsic properties of the antigen determine the character of immune reactivity for this method of immunization and that specific combination of innate and adaptive immune cytokine genes can increase the magnitude of the response to DNA vaccines.     

IL-12 or IL-4 prime human NK cells to mediate functionally divergent interactions with dendritic cells or tumors

In the course of inflammatory responses in peripheral tissues, NK cells may be exposed to cytokines such as IL-12 and IL-4 released by other cell types that may influence their functional activities. In the present study we comparatively analyzed purified human peripheral blood NK cells that had been exposed to either IL-12 or IL-4 during short (overnight) incubation. We show that although IL-12-cultured NK cells produced abundant IFN-gamma, TNF-alpha, and GM-CSF in response to stimuli acting on the NKp46-activating receptor, IL-4-cultured NK cells did not release detectable levels of these cytokines. In contrast, IL-4-cultured NK cells produced significant levels of TNF-alpha and GM-CSF only when stimulated with PMA and ionomycin. In no instance could the production of IL-5 and IL-13 be detected. Importantly, IL-12-cultured, but not IL-4-cultured, NK cells displayed strong cytolytic activity against various tumor cells or immature dendritic cells (DCs). Moreover, only NK cells that had been cultured in IL-12 were able to induce substantial DC maturation. Our data suggest that NK cells exposed to IL-12 for a time interval compatible with in vivo responses may favor the selection of appropriate mature DCs for subsequent Th1 cell priming in secondary lymphoid organs. On the contrary, NK cells exposed to IL-4 do not exert DC selection, may impair efficient Th1 priming, and favor either tolerogenic or Th2-type responses.     

Combinations of the cytokines IL-12, IL-2 and IFN-alpha significantly augment whereas the cytokine IL-4 suppresses the cytokine-induced antibody-dependent cellular cytotoxicity of monoclonal antibodies 17-1A and BR55-2

Since some cytokines effectively enhance the cytotoxicity of monoclonal antibodies, we investigated whether a combination of cytokines can augment the antibody-dependent cellular cytotoxicity (ADCC) of monoclonal antibodies 17-1A and BR55-2 against the colorectal carcinoma cell line HT29. Since monocytes/macrophages are important effector cells for ADCC, we used a new flow cytometric cytotoxicity assay, which allows the analysis of long-term-ADCC exerted by these cells. In our previous studies with peripheral blood mononuclear cells from normal donors, we found that IL-2, IL-12 and IFN-alpha increase ADCC. Therefore, we examined whether combination of these three cytokines with IL-2, IL-4, IL-6, IL-10, IL-12, IFN-alpha, IFN-gamma, GM-CSF, M-CSF and TNF-alpha may yield higher ADCC than obtained by the application of single cytokines. Indeed, we found that the combinations IL-2/IFN-alpha, IL-2/IL-12 and IL-12/IFN-alpha potentiated ADCC. Interestingly, the ineffective single cytokines TNF-alpha and GM-CSF in the combinations IL-2/TNF-alpha, IFN-alpha/TNF-alpha and IFN-alpha/GM-CSF also proved to enhance ADCC. In contrast, IL-4 significantly suppressed the IL-2, IL-12 and IFN-alpha-induced ADCC. In addition, the immunosuppressive cytokine IL-10 in higher concentrations significantly suppressed the IL-12-induced-ADCC. Our results may be useful to find combinations of cytokines and mAb for the treatment of cancer.     

T-cell immune responses against Env from CRF12_BF and subtype B HIV-1 show high clade-specificity that can be overridden by multiclade immunizations

Background

The extreme genetic diversity of the human immunodeficiency virus type 1 (HIV-1) poses a daunting challenge to the generation of an effective AIDS vaccine. In Argentina, the epidemic is characterized by the high prevalence of infections caused by subtype B and BF variants. The aim of this study was to characterize in mice the immunogenic and antigenic properties of the Env protein from CRF12_BF in comparison with clade B, employing prime-boost schemes with the combination of recombinant DNA and vaccinia virus (VV) vectors.

Methodology/Principal Findings

As determined by ELISPOT from splenocytes of animals immunized with either EnvBF or EnvB antigens, the majority of the cellular responses to Env were found to be clade-specific. A detailed peptide mapping of the responses reveal that when there is cross-reactivity, there are no amino acid changes in the peptide sequence or were minimal and located at the peptide ends. In those cases, analysis of T cell polifunctionality and affinity indicated no differences with respect to the cellular responses found against the original homologous sequence.Significantly, application of a mixed immunization combining both clades (B and BF) induced a broader cellular response, in which the majority of the peptides targeted after the single clade vaccinations generated a positive response. In this group we could also find significant cellular and humoral responses against the whole gp120 protein from subtype B.

Conclusions/Significance

This work has characterized for the first time the immunogenic peptides of certain EnvBF regions, involved in T cell responses. It provides evidence that to improve immune responses to HIV there is a need to combine Env antigens from different clades, highlighting the convenience of the inclusion of BF antigens in future vaccines for geographic regions where these HIV variants circulate.     

HIV-1-specific CTL responses primed in vitro by blood-derived dendritic cells and Th1-biasing cytokines

Vaccine strategies designed to elicit strong cell-mediated immune responses to HIV Ags are likely to lead to protective immunity against HIV infection. Dendritic cells (DC) are the most potent APCs capable of priming both MHC class I- and II-restricted, Ag-specific T cell responses. Utilizing a system in which cultured DC from HIV-seronegative donors were used as APC to present HIV-1 Ags to autologous T cells in vitro, the strength and specificity of primary HIV-specific CTL responses generated to exogenous HIV-1 Nef protein as well as intracellularly expressed nef transgene product were investigated. DC expressing the nef gene were able to stimulate Nef-specific CTL, with T cells from several donors recognizing more than one epitope restricted by a single HLA molecule. Primary Nef-specific CTL responses were also generated in vitro using DC pulsed with Nef protein. T cells primed with Nef-expressing DC (via protein or transgene) were able to lyse MHC class I-matched target cells pulsed with defined Nef epitope peptides as well as newly identified peptide epitopes. The addition of Th1-biasing cytokines IL-12 or IFN-alpha, during priming with Nef-expressing DC, enhanced the Nef-specific CTL responses generated using either Ag-loading approach. These results suggest that this in vitro vaccine model may be useful in identifying immunogenic epitopes as vaccine targets and in evaluating the effects of cytokines and other adjuvants on Ag-specific T cell induction. Successful approaches may provide information important to the development of prophylactic HIV vaccines and are envisioned to be readily translated into clinical DC-based therapeutic vaccines for HIV-1.     

Stimulation of IL-12 secretion by GM-CSF in advanced cancer patients

In addition to its efficacy in the treatment of chemotherapy-induced neutropenia, recent evidence would suggest that GM-CSF may have immunomodulatory effects on anticancer immunity. In particular, GM-CSF has been proven to promote dendritic cell maturation, with following potential stimulation of the anticancer cytokine, IL-12. Unfortunately, at present there are only few and controversial results on GM-CSF effects on IL-12 secretion in cancer patients. This preliminary study was performed to evaluate IL-12 response to an acute injection of GM-CSF in human neoplasms. The study included 20 advanced cancer patients, who received GM-CSF for chemotherapy-induced neutropenia. GM-CSF was injected at 3 micrograms/kg at 8.00 A.M., and venous blood samples were drawn before GM-CSF, and at 4, 8, 12 and 24 hours after its injection. Serum levels of IL-12 were measured by an enzyme immunoassay. High basal levels of IL-12 were seen in 8/20 patients. In patients with abnormally high pretreatment levels of IL-12, no significant change occurred in IL-12 mean serum concentration after GM-CSF administration. In contrast, patients with normal baseline levels of IL-12 showed a significant increase in IL-12 mean concentrations in response to GM-CSF, with a peak after 12 hours. This preliminary study seems to show that GM-CSF may acutely stimulate IL-12 secretion in cancer patients. Further studies are required to evaluate the effects of chronic GM-CSF administration, and the impact of GM-CSF-induced secretion of IL-12 on the efficacy of the immunotherapies of cancer with cytokines, such as IL-2. In any case, this study would justify further research in the emerging oncological applications of GM-CSF as an immunomodulatory agent on host anticancer defenses.     

The combination of DNA vectors expressing IL-12 + IL-18 elicits high protective immune response against cutaneous leishmaniasis after priming with DNA-p36/LACK and the cytokines,followed by a booster with a vaccinia virus recombinant expressing p36/LACK

Protocols of immunization based on the DNA prime/vaccinia virus (VV) boost regime with recombinants expressing relevant antigens have been shown to elicit protection against a variety of pathogens in animal model systems, and various phase I clinical trials have been initiated with this vaccination approach. We have previously shown that mice immunized with a DNA vector expressing p36/LACK of Leishmania infantum followed by a booster with VVp36/LACK induced significant protection against Leishmania major infection. To further improve this protocol of immunization, here we investigated whether the cytokines interleukin-12 (IL-12) and IL-18 could enhance protection against L. major infection in BALB/c mice. We found that priming with DNA vectors expressing p36/LACK and either IL-12 or IL-18, followed by a booster with a VV recombinant expressing the same L. infantum LACK antigen, elicit a higher cellular immune response than by using the same protocol in the absence of the cytokines. The cytokine IL-12 triggered a higher number of IFN-gamma-secreting cells specific for p36 protein than IL-18. When immunized animals were challenged with promastigotes, the highest protection against L. major infection was observed in animals primed with DNAp36 + DNA IL-12 + DNA IL-18 and boosted with VVp36. This protection correlated with a Th1 type of immune response. Our findings revealed that in prime/booster protocols, co-expressing IL-12 and IL-18 during priming is an efficient approach to protect against leishmaniasis. This combined prime/booster immunization regime could have wide use in fighting against parasitic and other infectious diseases.     

Vaccines based on Nef and on Nef/DeltaV2 Env

Modified vaccinia virus Ankara (MVA) is a potent vaccine vector, which proved its safety, immunogenity and efficacy in preclinical and clinical studies. The rational for the development of a vaccine against HIV based on the regulatory protein Nef delivered by MVA combined with a V2-deleted Env protein is discussed.     

Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization.

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope +/- cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1alpha plus IL-18, IL-1alpha plus IL-12, and IL-1alpha plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60-71% peptide-specific (51)Cr release). Peak H-2D(d)-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8(+) PBMC. Nasal immunization with HIV immunogen and IL-1alpha, IL-12, and GM-CSF also induced Ag-specific IFN-gamma-secreting cells in the draining cervical lymph node and the lung. The use of IL-1alpha, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1alpha, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.     

Multiprotein genetic vaccine in the SIV-Macaca animal model: a promising approach to generate sterilizing immunity to HIV infection

BACKGROUND: Vaccine combining structural and regulatory proteins is an emerging approach to develop an HIV/AIDS vaccine and therefore, the immunogenicity and efficacy of two regimens of immunization combining structural (Gag/Pol, Env) and regulatory (Rev, Tat, Nef) Simian immunodeficiency virus (SIV) proteins were compared in cynomolgus monkeys. METHODS: Monkeys were immunized with Modified Vaccine Ankara vector (MVA-J5) (protocol 1) or with DNA, Semliki forest virus and MVA vectors (DNA/SFV/MVA) (protocol 2). At week 32, all monkeys were challenge intravenously (protocol 1) or intrarectally (protocol 2) with 50 MID(50) of SIVmac251. Humoral, proliferative responses and in particular in protocol 2, the frequency of IFN-gamma producing cells, were measured in all monkeys before and after the challenge. RESULTS: Both vaccine regimens elicited humoral and proliferative responses but failed to induce neutralizing antibodies. Upon intravenous challenge, two out of three MVA-J5 vaccinated monkeys exhibited a long-term control of the viral replication whereas DNA/SFV/MVA vaccine abrogated the virus replication up to undetectable level in three out of four vaccinated monkeys. A major contribution to this vaccine effect appeared to be the IFN-gamma/ELISPOT responses to vaccine antigens (Gag, Rev Tat and Nef). CONCLUSIONS: These results, indicate that multiprotein heterologous prime-boost vaccination can induce a robust vaccine-induced immunity able to abrogate virus replication.     

Comparative affects of plasmid-encoded interleukin 12 and interleukin 18 on the protective efficacy of DNA vaccination against Mycobacterium tuberculosis

Protective immunity against Mycobacterium tuberculosis infection requires the induction and maintenance of mycobacteria-specific, IFN-gamma-secreting CD4+ and CD8+ T lymphocytes. The development of Th1-like T cells is promoted by the early secretion and synergistic action of interleukin (IL)-12 and IL-18. This study compares the effects of plasmid-encoded IL-12 and IL-18 on the immunogenicity and protective efficacy of a DNA vaccine expressing the M. tuberculosis-secreted protein antigen 85B (DNA-85B). Co-immunization with either IL-12- or IL-18-expressing plasmids augmented the IFN-gamma-secreting T-cell response, and the maximum effect was observed with plasmids encoding both cytokines. Further the IL-12, but not the IL-18-expressing plasmid, significantly increased the protective efficacy of DNA-85B against pulmonary M. tuberculosis infection. Therefore co-administration of plasmid-encoded cytokines provides a potential method for optimizing the protective efficacy of DNA vaccination against tuberculosis.     

Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors

The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-gamma). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-gamma, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection.     

Mapping HIV-1 vaccine induced T-cell responses: bias towards less-conserved regions and potential impact on vaccine efficacy in the Step study

T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. Trial registration: ClinicalTrials.gov NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.     

Magnitude, breadth, and functional profile of T-cell responses during human immunodeficiency virus primary infection with B and BF viral variants

The molecular pattern of the human immunodeficiency virus (HIV) epidemic in Argentina provides an appropriate scenario to study cellular immune responses in patients with non-clade B infection. We aimed to map T-cell responses in patients infected with BF recombinant variants and compare them with those of clade B patients. Sixteen recently infected patients were enrolled and grouped by viral subtype. Nef-specific responses were evaluated with a peptide matrix-based gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay using B and BF overlapping peptides. Cross-clade and clade-specific responses were found. A correlation between B versus BF Nef-specific responses was identified. Detailed analysis at the single-peptide level revealed that BF patients show a narrower response but greater magnitude. Nef immunodominant responses agreed with previous publications, although the B loop was targeted at an unexpectedly high frequency. The putative HLA allele(s) restricting each positive response was determined. Single-peptide level screening with two different peptide sets uncovered discordant responses (mostly caused by peptide offsetting) and allowed detection of increased breadth. Positive responses identified by ELISPOT assay were further studied by intracellular cytokine staining. These were almost exclusively mediated by CD8 T cells. Characterization of concordant responses revealed that cells show distinct functional profiles, depending on the peptide presented. Last, quality (in terms of polyfunctionality) of T cells was associated with better viral replication containment. Overall, interclade differences in the frequency of epitopes recognized, structural domains targeted, and magnitude of responses were identified. Screening T-cell responses with multiple sets increased sensitivity. Further support for the notion of polyfunctional CD8⁺ T-cell requirement to better control viral replication is also provided.     

Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection

The characterization of the cross-reactive, or heterologous, neutralizing antibody responses developed during human immunodeficiency virus type 1 (HIV-1) infection and the identification of factors associated with their generation are relevant to the development of an HIV vaccine. We report that in healthy HIV-positive, antiretroviral-naïve subjects, the breadth of plasma heterologous neutralizing antibody responses correlates with the time since infection, plasma viremia levels, and the binding avidity of anti-Env antibodies. Anti-CD4-binding site antibodies are responsible for the exceptionally broad cross-neutralizing antibody responses recorded only in rare plasma samples. However, in most cases examined, antibodies to the variable regions and to the CD4-binding site of Env modestly contributed in defining the overall breadth of these responses. Plasmas with broad cross-neutralizing antibody responses were identified that targeted the gp120 subunit, but their precise epitopes mapped outside the variable regions and the CD4-binding site. Finally, although several plasmas were identified with cross-neutralizing antibody responses that were not directed against gp120, only one plasma with a moderate breadth of heterologous neutralizing antibody responses contained cross-reactive neutralizing antibodies against the 4E10 epitope, which is within the gp41 transmembrane subunit. Overall, our study indicates that more than one pathway leads to the development of broad cross-reactive neutralizing antibodies during HIV infection and that the virus continuously escapes their action.     

Enhancement by IL-12 of the cytolytic T lymphocyte (CTL) response of mice immunized with tumor-specific peptides in an adjuvant containing QS21 and MPL.

Immunization of cancer patients with tumor-specific antigenic peptides is currently being tested in several clinical studies. We have examined the induction of CTL responses in mice after various modalities of peptide vaccination, to explore protocols that could be applied to humans. Our first model antigen was P198, which results from a point mutation in a normal gene. While two immunizations with peptide P198 in SBAS-1c adjuvant induced measurable CTL responses in less than 10% of DBA/2 mice, the addition of IL-12 to the peptide adjuvant mixture resulted in high CTL responses in nearly all mice. This strong enhancing effect of IL-12 was observed with 1,000 and 300 units and decreased gradually as the doses were reduced to 30 units. When IL-12 was replaced by other cytokines acting on T cells or antigen-presenting cells, such as IFN-gamma, IL-2, IL-6, IL-7, GM-CSF or MCP-3, no significant enhancing effect was observed. The same effect of IL-12 was obtained with peptide P1A, which is a major tumor-specific antigen of mastocytoma P815 and is encoded by a gene that is specifically activated in tumors.