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Upon the introduction of the combination antiretroviral therapy (cART), HIV infection has become a chronic disease. However, cART is unable to eradicate the virus and fails to restore the CD4 counts in about 30% of the treated individuals. Furthermore, treatment is life-long, and it does not protect from morbidities typically observed in the elderly. Therapeutic vaccines represent the most cost-effective intervention to intensify or replace cART.Here, we briefly discuss the obstacles to the development and evaluation of the efficacy of therapeutic vaccines and review recent approaches evaluated in clinical trials.Although vaccines were generally safe and immunogenic, evidence of efficacy was negligible or marginal in most trials. A notable exception is the therapeutic Tat vaccine approach showing promising results of cART intensification, with CD4 T-cell increase and proviral load reduction beyond those afforded by cART alone. Rationale and evidence in support of choosing Tat as the vaccine target are thoroughly discussed.

Despite over 30 years of enormous effort and progress in the field, no preventative and/or therapeutic vaccines against human immunodeficiency virus (HIV) are available. Here, we briefly summarize the vaccine strategies and vaccine candidates that in recent years advanced to efficacy trials with mostly unsatisfactory results. Next, we discuss a novel and somewhat contrarian approach based on biological and epidemiological evidence, which led us to choose the HIV protein Tat for the development of preventive and therapeutic HIV vaccines. Toward this goal, we review here the role of Tat in the virus life cycle as well as experimental and epidemiological evidence supporting its key role in the natural history of HIV infection and comorbidities. We then discuss the preclinical and clinical development of a Tat therapeutic vaccine, which, by improving the functionality and homeostasis of the immune system and by reducing the viral reservoir in virologically suppressed vaccinees, helps to establish key determinants for intensification of combination antiretroviral therapy (cART) and a functional cure. Future developments and potential applications of the Tat therapeutic vaccine are also discussed, as well as the rationale for its use in preventative strategies. We hope this contribution will lead to a reconsideration of the current paradigms for the development of HIV/AIDS vaccines, with a focus on targeting of viral proteins with key roles in HIV pathogenesis.

Introduction: Although successful at suppressing HIV replication, combination antiretroviral therapy (cART) only partially restores immune functions and fails to reduce the latent HIV reservoir, th...

Objective:HIV infection is characterized by several immune dysfunctions, such as chronic activation of the immune system, premature aging and loss of CD4(+) T cells, in particular within the naive compartment. The Tat protein of HIV is released extracellularly and enters neighboring cells affecting their functionality, for instance impacting on CD8(+) T-cell programs and activity. As the presence and/or induction of anti-Tat immune responses is associated with reduced T-cell dysfunction and CD4(+) T-cell loss, we investigated whether Tat impacts human resting or activated CD4(+) T cells.Methods:Purified CD4(+) T cells were activated by T cell receptor engagement in the presence or absence of Tat. Cytokine production, surface phenotype and expression of transcription factors important for T-cell programing were measured. Purified naive CD4(+) T cells were cultured in nonpolarizing conditions in the presence or absence of Tat and their proliferation and differentiation was evaluated.Results:Tat favors the secretion of IL2, IFN and TNF in CD4(+) T cells, as well as the upregulation of T-bet and Eomes expression. Naive CD4(+) T cells cultured in the presence of Tat showed enhanced expansion and differentiation toward memory phenotype, showing in particular recruitment into the effector memory T-cell pool.Conclusion:Tat affects the programing and functionality of CD4(+) T lymphocytes favoring the differentiation of naive CD4(+) T cells.

Classical approaches aimed at targeting the HIV-1 envelope as well as other structural viral proteins have largely failed. The HIV-1 transactivator of transcription (Tat) is a key HIV virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. Notably, anti-Tat Abs are uncommon in natural infection and, when present, correlate with the asymptomatic state and lead to lower or no disease progression. Hence, targeting Tat represents a pathogenesis-driven intervention.Here, we review the rationale and the translational development of a therapeutic vaccine targeting the Tat protein. Preclinical and Phase I studies, Phase II trials with Tat in anti-Tat Ab-negative, virologically suppressed highly active antiretroviral therapy-treated subjects in Italy and South Africa were conducted. The results indicate that Tat-induced immune responses are necessary to restore immune homeostasis, to block the replenishment and to reduce the size of the viral reservoir. Additionally, they may help in establishing key parameters for highly active antiretroviral therapy intensification and a functional cure.We propose the therapeutic setting as the most feasible to speed up the testing and comparison of preventative vaccine candidates, as the distinction lies in the use of the vaccine in uninfected versus infected subjects and not in the vaccine formulation.

Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/TatΔ51–59virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA) of A/WSN/33 virus. The TatΔ51–59insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replicationin vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/TatΔ51–59virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines.

Background Although combined antiretroviral therapy (cART) has saved millions of lives, it is incapable of full immune reconstitution and virus eradication. The transactivator of transcription (Tat) protein is a key human immunodeficiency virus (HIV) virulence factor required for virus replication and transmission. Tat is expressed and released extracellularly by infected cells also under cART and in this form induces immune dysregulation, and promotes virus reactivation, entry and spreading. Of note, anti-Tat antibodies are rare in natural infection and, when present, correlate with asymptomatic state and reduced disease progression. This suggested that induction of anti-Tat antibodies represents a pathogenesis-driven intervention to block progression and to intensify cART. Indeed Tat-based vaccination was safe, immunogenic and capable of immune restoration in an open-label, randomized phase II clinical trial conducted in 168 cART-treated volunteers in Italy. To assess whether B-clade Tat immunization would be effective also in patients with different genetic background and infecting virus, a phase II trial was conducted in South Africa. Methods The ISS T-003 was a 48-week randomised, double-blinded, placebo-controlled trial to evaluate immunogenicity (primary endpoint) and safety (secondary endpoint) of B-clade Tat (30 μg) given intradermally, three times at 4-week intervals, in 200 HIV-infected adults on effective cART (randomised 1:1) with CD4+ T-cell counts ≥200 cells/µL. Study outcomes also included cross-clade anti-Tat antibodies, neutralization, CD4+ T-cell counts and therapy compliance. Results Immunization was safe and well-tolerated and induced durable, high titers anti-Tat B-clade antibodies in 97 % vaccinees. Anti-Tat antibodies were cross-clade (all vaccinees tested) and neutralized Tat-mediated entry of oligomeric B-clade and C-clade envelope in dendritic cells (24 participants tested). Anti-Tat antibody titers correlated positively with neutralization. Tat vaccination increased CD4+ T-cell numbers (all participants tested), particularly when baseline levels were still low after years of therapy, and this had a positive correlation with HIV neutralization. Finally, in cART non-compliant patients (24 participants), vaccination contained viral load rebound and maintained CD4+ T-cell numbers over study entry levels as compared to placebo. Conclusions The data indicate that Tat vaccination can restore the immune system and induces cross-clade neutralizing anti-Tat antibodies in patients with different genetic backgrounds and infecting viruses, supporting the conduct of phase III studies in South Africa. Trial registration ClinicalTrials.gov NCT01513135, 01/23/2012 Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0261-1) contains supplementary material, which is available to authorized users.

A workshop entitled: “Revisiting HIV inactivation, elite controllers, immunogenetics and new strategies for developing HIV vaccines” took place during a Eurovaccine Conference held in Rome in June 2016. The purpose of this workshop was to revisit old and new concepts and strategies in HIV vaccinology in the light of novel, and sometimes unexpected, data from recent preventative and therapeutic vaccine approaches that could guide future vaccine research. Panelists were asked to respond to five questions regarding key points and critical issues and problems in current HIV/AIDS vaccine research. Their responses are summarized.

Mauritian cynomolgus macaques (MCM) are widely used in human immunodeficiency virus research because of their restricted major histocompatibility complex (MHC) diversity which provides the opportunity to address the influence of host factors on vaccine studies. We herein report the impact of MHC haplotype on the outcome of 21 MCM infections with the CCR5-tropic simian/human immunodeficiency virus (SHIV)(SF162P4cy). MCM were susceptible to SHIV(SF162P4cy) infection as shown by viremia and loss of CD4+ T cells. A significant association between haplotype M7 (class IA, IB, II) and persistent viremia was observed in chronic phase, whereas recombinant class IA haplotype was associated with a reduction of viral RNA during acute infection. Class IB M4 haplotype displayed significantly lower acute phase provirus copy numbers. In addition, statistical analysis indicated a detrimental effect of haplotype M4 (class IA, IB) on the course of infection as indicated by lower CD4+ T-cell levels during chronic infection. A decrease in post-acute phase CD4+ T-cell numbers was also observed in haplotype M2 animals. This is the first report that documents the effects of host MHC class I and II molecules on the SHIV(SF162P4cy) infection in MCM, particularly with regard to the association between recombinant class IA, M4, and M7 haplotypes and the dynamic of viral replication and level of CD4+ T cells.

HIV native Tat and V2 loop-deleted Env (EnvΔV2) proteins already proved safe and immunogenic in phase I clinical testing as single vaccine components. Further, a phase II vaccine trial with Tat showed intensification of the therapeutic effects of HAART in successfully treated HIV-infected individuals. Here a pilot study assessed the immunogenicity and protective efficacy of an HIV/AIDS vaccine based on the combination of Tat and EnvΔV2 proteins in cynomolgus macaques against homologous intrarectal challenge with 35 MID(50) (monkey infectious dose 50) of an R5 simian-human immunodeficiency virus (SHIV(SF162P4cy)). Upon challenge, three of four macaques immunized with Tat and EnvΔV2, and two of three monkeys immunized with EnvΔV2 alone were protected from infection. In contrast, all three control animals, which had been either administered with the adjuvants only or left untreated, and an additional monkey immunized with Tat alone became systemically infected. Protection of the macaques vaccinated with EnvΔV2 or Tat/EnvΔV2 correlated with higher peak titers of pre-challenge neutralizing antibodies obtained during the immunization period (between 70 and 3 weeks before challenge) and with anti-Env V3 loop binding antibodies assessed 3 weeks before challenge. Compared to EnvΔV2 alone, the Tat and EnvΔV2 combined vaccine elicited faster antibody responses (IgM) with a trend, early in the vaccination schedule, after the second immunization including EnvΔV2, towards broader anti-Env IgG epitope specificity and a higher ratio of neutralizing to Env-binding antibody titers. As the number of immunizations increased, vaccination with EnvΔV2 approached the immune response assessed after two inocula with the Tat/EnvΔV2 combined vaccine, even though some differences remained between groups, as indicated by anti-Env IgG epitope mapping. In fact, three weeks before challenge, plasma IgG of animals in the EnvΔV2 group showed a trend towards stronger specificity for the V1 loop and V5 loop-C5 regions of Env, whereas the Tat/EnvΔV2 group displayed an overall higher reactivity for epitopes within the Env V3 loop throughout the immunization period. Although differences in terms of protection rate were not found between the EnvΔV2 or Tat/EnvΔV2 vaccination groups in this pilot study, vaccination with Tat/EnvΔV2 appeared to accelerate the induction of potentially protective antibody responses to Env. In particular, antibodies to the Env V3 loop, whose levels at pre-challenge correlated with protection, were already higher early in the vaccination schedule in monkeys immunized with Tat/EnvΔV2 as compared to EnvΔV2 alone. Further studies including larger vaccination groups and fewer immunizations with these two vaccine candidates are needed to confirm these findings and to assess whether the Tat/EnvΔV2 vaccine may afford superior protection against infection.

The effects of the challenge dose and major histocompatibility complex (MHC) class IB alleles were analyzed in 112 Mauritian cynomolgus monkeys vaccinated ( n = 67) or not vaccinated ( n = 45) with Tat and challenged with simian/human immunodeficiency virus (SHIV) 89.6P cy243. In the controls, the challenge dose (10 to 20 50% monkey infectious doses [MID 50 ]) or MHC did not affect susceptibility to infection, peak viral load, or acute CD4 T-cell loss, whereas in the chronic phase of infection, the H1 haplotype correlated with a high viral load ( P = 0.0280) and CD4 loss ( P = 0.0343). Vaccination reduced the rate of infection acquisition at 10 MID 50 ( P < 0.0001), and contained acute CD4 loss at 15 MID 50 ( P = 0.0099). Haplotypes H2 and H6 were correlated with increased susceptibility ( P = 0.0199) and resistance ( P = 0.0087) to infection, respectively. Vaccination also contained CD4 depletion ( P = 0.0391) during chronic infection, independently of the challenge dose or haplotype.

Protection afforded by HIV Tat-based vaccines has differed in Indian rhesus and Mauritian cynomolgus macaques. We evaluated native Tat and Ad-HIVtat priming/Tat-boosting regimens in both species. Both vaccines were immunogenic. Only the Ad-tat regimen modestly reduced acute viremia in rhesus macaques after SHIV(89.6P) challenge. Confounding variables uncovered in Mauritian macaques included significant associations of susceptibility to infection with MHC class IB and class II H2 and H5 haplotypes, and resistance to infection with class IB haplotypes H3 and H6. Although protection here was limited, Tat-based vaccines incorporating other HIV components have shown greater efficacy. Combination strategies should be further explored.

We have previously shown that the biologically active Tat protein targets and efficiently enters dendritic cells, and increases the proteolytic activities of the immunoproteasome, thereby favoring the generation and presentation of the subdominant MHC-I binding CTL epitopes of heterologous antigens. In the present study, we demonstrate that Tat broadens in vivo epitope-specific T cell responses directed to heterologous antigens including HIV structural proteins. Specifically, co-immunization of mice with OVA and Tat proteins induces CTL responses against subdominant and cryptic OVA-derived epitopes, which are not detected in mice vaccinated with OVA alone. Similarly, mice vaccinated with the HIV-1 Gag, Env or V2-deleted Env antigens in combination with Tat show Th1-type and CTL responses directed to a larger number of T cell epitopes, as compared to mice vaccinated with these proteins in absence of Tat. In contrast, Tat did not affect Th2-type responses to these structural HIV proteins. These results indicate that Tat is not only an antigen but also a novel Th1-type adjuvant capable of broadening in vivo the spectrum of epitopes recognized by T cells, and suggest that Tat can be considered an optimal co-antigen in the development of novel vaccination strategies against AIDS.

Simian-human immunodeficiency virus (SHIV) 89.6P is considered to be one of the most pathogenic chimeric viruses in rhesus macaques. However, when crossing from one to another species of monkeys the pathogenicity of this virus may be affected. By using SHIV-89.6P(cy243), a virus obtained by passaging SHIV-89.6P in cynomolgus macaques, we investigated the dynamics of viral replication and the impact of the inoculum size (from 10 up to 50 monkey infectious dose) on the progression of the infection in 22 cynomolgus macaques. SHIV-89.6P(cy243 )caused massive depletion of CD4+ T-cells within 4 weeks of the inoculum, followed by an irreversible immune deficiency in a high proportion of the infected monkeys. This study demonstrates that SHIV-89.6P(cy243) is pathogenic in cynomolgus macaques and that the dynamics of the viral replication and the rate of clinical progression depend on the size of the inoculum. Our findings provide unique and relevant data, particularly with regard to the value of the in vivo titration used to select the most appropriate infectious dose to study the "virus-host" interplay.

Pre-clinical HIV-1 vaccine protocols, using multiple vaccine modalities and a potent adjuvant were assessed for vaccine efficacy in an experimental HIV-1 challenge model. C57Bl/6 mice were immunized with DNA plasmids encoding HIV-1 gp140, Gag and Tat alone or in combination with the corresponding recombinant proteins formulated in the adjuvant MF59. HIV-1 DNA alone or a DNA prime protein boost schedule resulted in complete protection against challenge with HIV-1/MuLV-infected murine cells. Although HIV-1 protein immunization in combination with MF59 resulted in partial protection, the DNA priming seemed to be crucial for obtaining full protection against the challenge. It is likely that the partial protection seen after immunization with protein alone is, to a certain extent, due to effects of the adjuvant since some animals that received the adjuvant MF59 alone were protected from the challenge. For the most part, antigen-specific cell-mediated immune responses as detected in the spleen (in contrast to responses detected in peripheral blood) of immunized animals appeared to be associated with protection in this study.

We previously demonstrated that replication-competent adenovirus (Ad)-simian immunodeficiency virus (SIV) recombinant prime/protein boost regimens elicit potent immunogenicity and strong, durable protection of rhesus macaques against SIVmac251. Additionally, native Tat vaccines have conferred strong protection against simian/human immunodeficiency virus SHIV89.6Pchallenge of cynomolgus monkeys, while native, inactivated, or vectored Tat vaccines have failed to elicit similar protective efficacy in rhesus macaques. Here we asked if priming rhesus macaques with replicating Ad-human immunodeficiency virus (HIV)tatand boosting with the Tat protein would elicit protection against SHIV89.6P. We also evaluated a Tat/Env regimen, adding an Ad-HIVenvrecombinant and envelope protein boost to test whether envelope antibodies would augment acute-phase protection. Further, expecting cellular immunity to enhance chronic viremia control, we tested a multigenic group: Ad-HIVtat, -HIVenv, -SIVgag, and -SIVnefrecombinants and Tat, Env, and Nef proteins. All regimens were immunogenic. A hierarchy was observed in enzyme-linked immunospot responses (with the strongest response for Env, followed by Gag, followed by Nef, followed by Tat) and antibody titers (with the highest titer for Env, followed by Tat, followed by Nef, followed by Gag). Following intravenous SHIV89.6Pchallenge, all macaques became infected. Compared to controls, no protection was seen in the Tat-only group, confirming previous reports for rhesus macaques. However, the multigenic group blunted acute viremia by approximately 1 log (P= 0.017), and both the multigenic and Tat/Env groups reduced chronic viremia by 3 and 4 logs, respectively, compared to controls (multigenic,P= 0.0003; Tat/Env,P< 0.0001). The strikingly greater reduction in the Tat/Env group than in the multigenic group (P= 0.014) was correlated with Tat and Env binding antibodies. Since prechallenge anti-Env antibodies lacked SHIV89.6P-neutralizing activity, other functional anti-Env and anti-Tat activities are under investigation, as is a possible synergy between the Tat and Env immunogens.

Mucosal HSV infection remains a public health issue in developing and developed world. However, an effective vaccine is still missing, partly because of the incomplete knowledge of correlates of protection. In this study we have investigated the kinetics and quality of immunity elicited by an attenuated HSV1 vector expressing the immunomodulatory Tat protein of HIV-1 (HSV1-Tat). Animals were immunized by intravaginal (IVag) or intradermal (ID) route with HSV1-Tat or with a control HSV1 vector expressing the LacZ gene (HSV1-LacZ) and immune responses were characterized in different anatomical districts. IVag immunization with HSV1-Tat enhanced both expansion and memory phases of HSV-specific immunodominant CD8 responses at systemic, but not local, level and induced short- and long-term protection against mucosal challenge. Conversely, ID immunization with HSV1-Tat favored HSV-subdominant CD8 responses, which protected mice only at early time points after immunization. IVag immunization, in particular with HSV1-Tat, compared to ID immunization, induced the differentiation of CD8(+) T lymphocytes into short-lived effector (SLEC) and effector memory (Tem) cells, generating more robust recall responses associated with increased control of virus replication. Notably, systemic SLEC and Tem contributed to generate protective local secondary responses, demonstrating their importance for mucosal control of HSV. Finally, IgG responses were observed mostly in IVag HSV1-Tat immunized animals, although seemed dispensable for protection, which occurred even in few IgG negative mice. Thus, HSV1 vectors expressing Tat induce protective anti-HSV1 immune responses.

Background The phase II multicenter, randomized, open label, therapeutic trial (ISS T-002, Clinicaltrials.gov NCT00751595) was aimed at evaluating the immunogenicity and the safety of the biologically active HIV-1 Tat protein administered at 7.5 or 30 μg, given 3 or 5 times monthly, and at exploring immunological and virological disease biomarkers. The study duration was 48 weeks, however, vaccinees were followed until the last enrolled subject reached the 48 weeks. Reported are final data up to 144 weeks of follow-up. The ISS T-002 trial was conducted in 11 clinical centers in Italy on 168 HIV positive subjects under Highly Active Antiretroviral Therapy (HAART), anti-Tat Antibody (Ab) negative at baseline, with plasma viremia

Over the past 20 years most of the efforts in HIV vaccinedevelopment have focused on sterilizing immunity bytargeting the Envelope protein (Env). However, resultsfrom preclinical and clinical trials have been largelydisappointing [1–11]. Therefore, current vaccine strat-egies are not only aimed at preventing virus infection butalso at blocking virus replication and disease onset. Inparticular, the control of virus replication should provideprotection from disease development and reduce virustransmission, halting the HIV epidemic. This objectivemay be achieved by targeting virus regulatory genes,which are expressed early after infection, are essential forvirus replication and pathogenesis, and are moreconserved among HIV clades. This approach may beeffective for both preventive and therapeutic vaccinestrategies [12–68]. In this article we review thecharacteristics of Tat and why it was selected for use in avaccine.Wealsocitethelessonlearnedinthedevelopmentof this anti-Tat vaccine for use in human clinical trials.

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin–avidin–biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat. Balb/c mice were then immunized with 10g of soluble Tat in complete Freund’s adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5g soluble Tat in incomplete Freund’s adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies. © 2002 Elsevier Science Ltd. All rights reserved.

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.

Background: Tat is a key HIV-1 virulence factor, which plays pivotal roles in virus gene expression, replication, transmission and disease progression. After release, extracellular Tat accumulates in tissues and exerts effects on both the virus and the immune system, promoting immune activation and virus spreading while disabling the host immune defense. In particular, Tat binds Env spikes on virus particles forming a virus entry complex, which favors infection of dendritic cells and efficient transmission to T cells via RGD-binding integrins. Tat also shields the CCR5-binding sites of Env rendering ineffective virus neutralization by anti-Env antibodies (Abs). This is reversed by the anti-Tat Abs present in natural infection or induced by vaccination.Findings: Here we present the results of a cohort study, showing that the presence of anti-Tat Abs in asymptomatic and treatment-naïve HIV-infected subjects is associated with containment of CD4+ T-cell loss and viral load and with a delay of disease progression. In fact, no subjects with high anti-Tat Ab titers initiated antiretroviral therapy during the three years of follow-up. In contrast, no significant effects were seen for anti-Env and anti-Gag Abs. The increase of anti-Env Ab titers was associated with a reduced risk of starting therapy only in the presence of anti-Tat Abs, suggesting an effect of combined anti-Tat and anti-Env Abs on the Tat/Env virus entry complex and on virus neutralization.Conclusions: Anti-Tat immunity may help delay HIV disease progression, thus, targeting Tat may offer a novel therapeutic intervention to postpone antiretroviral treatment or to increase its efficacy. © 2014 Bellino et al.; licensee BioMed Central Ltd

Objective HIV infection is characterized by several immune dysfunctions of both CD8⁺ and CD4⁺ T cells as hyperactivation, impairment of functionality and expansion of memory T cells. CD8⁺ T-cell dysfunctions have been associated with increased expression of T-bet, Eomesdermin and pro-inflammatory cytokines, and with down-regulation of CD127. The HIV-1 trans-activator of transcription (Tat) protein, which is released by infected cells and detected in tissues of HIV-positive individuals, is known to contribute to the dysregulation of CD4⁺ T cells; however, its effects on CD8⁺ T cells have not been investigated. Thus, in this study, we sought to address whether Tat may affect CD8⁺ T-cell functionality and programming. Methods CD8⁺ T cells were activated by T-cell receptor engagement in the presence or absence of Tat. Cytokine production, killing capacity, surface phenotype and expression of transcription factors important for T-cell programming were evaluated. Results Tat favors the secretion of interleukin-2, interferon-γ and granzyme B in CD8⁺ T cells. Behind this functional modulation we observed that Tat increases the expression of T-bet, Eomesdermin, Blimp-1, Bcl-6 and Bcl-2 in activated but not in unstimulated CD8⁺ T lymphocytes. This effect is associated with the down-regulation of CD127 and the up-regulation of CD27. Conclusion Tat deeply alters the programming and functionality of CD8⁺ T lymphocytes.

Natural killer (NK) cell recognition and function in humans is regulated by multiple cell surface receptors. The "on" signal leading to NK cell triggering is primarily mediated by natural cytotoxicity receptors (NCR). Analysis of NK cells in primate animal models is of particular relevance because NK cells may play an essential role in host defenses against infections. We analyzed Macaca fascicularis PBMC and in vitro-derived NK cell populations and clones by cytofluorometry, using a wide panel of mAb, and by cytolytic activity assays. In addition, RT-PCR strategy and transient transfections were used to isolate M. fascicularis NCR. NCR-specific mAb reactivity (anti-NKp46 and anti-NKp30) was present on M. fascicularis PBMC and on NK cell cultures. Macaque NCR were functional in both redirected killing and in mAb-mediated masking assays. Cloning of macNKp46 and macNKp30 NCR homologous genes showed a high sequence similarity (86 % and 88 %, respectively) with their human counterparts. Attempts at identifying NKp44 surface reactivity and at cloning the macaque homologue were unsuccessful. NKp46 and NKp30 NCRs, but not NKp44, are highly conserved in M. fascicularis NK cells. This suggests the possibility of a staged appearance of the NCR during phylogenesis and provides a useful tool for the study of natural immunity correlates of protection in primate SIV/SHIV infection models.

Various simian immunodeficiency virus (SIV)sm/mac and simian/human immunodeficiency virus (SHIV) strains are used in different macaque species to study AIDS pathogenesis, as well as to evaluate candidate vaccine and anti-retroviral drugs efficacy. In this study we investigated the effect of route of infection, species of macaques and nature of virus stock on early plasma viral RNA load. We monitored the plasma RNA concentrations of 63 rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) infected with well-characterised virus stocks administered either by oral, rectal, vaginal or intravenous (i.v.) routes. In SIV(mac)-infected macaques, no significant difference in plasma RNA loads was observed between the rectal, oral and i.v. routes of infection. Cynomolgus macaques developed lower steady state SIV plasma RNA concentrations compared with rhesus macaques and no significant difference was observed between rectal and i.v. routes of infection. In SHIV(89.6p)-infected macaques, no difference between species or between route of infection was observed with this particular chimeric virus.

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.

Objective: To investigate the source of the expanded blood CD8+ subsets during an acute primary simian immunodeficiency virus (SIV) infection of macaques and the potential role of these cells in disease progression. Design and methods: The primary CD8+ lymphocytosis, which occurs at 1-2 weeks following infection with SIV smm/PBj-14' was examined in rhesus and cynomolgus macaques. Extensive subset analysis of the expanded blood CD8+ cell pool in a rhesus macaque was compared phenotypically with those in thymus, lymph nodes, spleen, ileum and lung washouts obtained at necropsy during blood lymphocytosis. The influence of the primary CD8+ cell expansion on disease progression was assessed at days 175-679 post-infection in long-term PBj-14 survivors staged according to immunological, virological and histopathological changes in their lymphoid organs. Result: The very rapid and transient blood lymphocytosis following infection consisted of two distinct CD45RA low , CD8+ and CD28-, lymphocyte function-associated antigen (LFA)-1 high , CD45RA high , CD8+ populations. These populations were present in low levels in thymus, lymph and spleen but were highly represented in mucosal tissues, such as lung washout, in which CD28- LFA-1 high CD45RA high CD8+ cells comprised 86% of CD8+ cells, and gut, which was predominantly CD45RA low CD28- CD8+ cells. A comparison of progressor and non-progressor PBj-14-infected rhesus and cynomolgus macaques also indicated that the existence or magnitude of a blood CD8+ lymphocytosis during the acute phase of infection did not by itself appear to influence or be predictive of disease progression. Conclusion: The marked blood CD8+ lymphocytosis observed during acute SIV infection did not result from expansion of virus-specific precursors in peripheral lymph node and did not appear to influence the rate of disease progression. The findings provide a novel explanation for the primary CD8+ cell lymphocytosis and invoke a mechanism whereby virus-induced cytokine/chemokine production in mucosal sites initiate the transient migration of a pre-existing CD8+ population into the blood from compartments such as lung and gut. Such results suggest that the magnitude of lymphocytosis may depend on the level of viral replication in mucosal tissues and the presence of other infections, for example, cytomegalovirus.

Decline in blood CD4+ lymphocytes during primary symptomatic infections with HIV is usually attributed to viral killing, and has not been considered in terms of altered lymphocyte migration and sequestration. We therefore sought to examine whether CD4+ cell loss from blood of macaques undergoing an acute primary SIV infection might be due to increased synthesis of cytokines, known to profoundly affect lymphocyte trafficking, rather than to direct lymphocyte destruction by virus. The findings indicate that rapid lymphocyte depletion following acute infection is not selective for CD4+ cells, correlates precisely with increased plasma IFN-gamma and tumor necrosis factor-alpha levels, and is reversible. CD4/CD8 ratios in lymph nodes with high viral burdens remain relatively unchanged despite lymphocyte loss from blood. Levels of cytokine mRNA measured in lymphoid organs reflect neither cytokine plasma levels nor their potential to induce sequestration. These results support a model of cytokine-induced lymphocyte extravasation to account for the acute HIV/SIV-induced CD4+ cell lymphopenia and raise questions regarding the extent to which altered lymphocyte migration plays a role in the gradual CD4+ cell depletion throughout infection.

The acquired immunodeficiency syndrome (AIDS) emerged in the human population in the summer of 1981. According to the latest United Nations estimates, worldwide over 33 million people are infected with human immunodeficiency virus (HIV) and the prevalence rates continue to rise globally. To control the alarming spread of HIV, an urgent need exists for developing a safe and effective vaccine that prevents individuals from becoming infected or progressing to disease. To be effective, an HIV/AIDS vaccine should induce broad and long-lasting humoral and cellular immune responses, at both mucosal and systemic level. However, the nature of protective immune responses remains largely elusive and this represents one of the major roadblocks preventing the development of an effective vaccine. Here we summarize our present understanding of the factors responsible for resistance to infection or control of progression to disease in human and monkey that may be relevant to vaccine development and briefly review recent approaches which are currently being tested in clinical trials. Finally, the rationale and the current status of novel strategies based on nonstructural HIV-1 proteins, such as Tat, Nef and Rev, used alone or in combination with modified structural HIV-1 Env proteins are discussed.

The acquired immunodeficiency syndrome (AIDS) emerged in the human population in the summer of 1981. According to the latest United Nations estimates, worldwide over 33 million people are infected with human immunodeficiency virus (HIV) and the prevalence rates continue to rise globally. To control the alarming spread of HIV, an urgent need exists for developing a safe and effective vaccine that prevents individuals from becoming infected or progressing to disease. To be effective, an HIV/AIDS vaccine should induce broad and long-lasting humoral and cellular immune responses, at both mucosal and systemic level. However, the nature of protective immune responses remains largely elusive and this represents one of the major roadblocks preventing the development of an effective vaccine. Here we summarize our present understanding of the factors responsible for resistance to infection or control of progression to disease in human and monkey that may be relevant to vaccine development and briefly review recent approaches which are currently being tested in clinical trials. Finally, the rationale and the current status of novel strategies based on nonstructural HIV-1 proteins, such as Tat, Nef and Rev, used alone or in combination with modified structural HIV-1 Env proteins are discussed.

The development of a vaccine against HIV/AIDS capable of inducing broad humoral and cellular responses at both systemic and mucosal sites, able to stop or reduce viral infection at the portal of entry, represents the only realistic way to control the infection caused by HIV world-wide. The promising results obtained with the HIV-1 Tat-based vaccines in preclinical and clinical settings, the evidence that a broad immunity against HIV correlates with reduced viral load or virus control, as well as the availability of novel gp140 V2-loop deleted HIV-1 Env (DeltaV2Env) immunogens capable of inducing cross-reactive neutralizing antibodies, have led to the design of new vaccine strategies based on the combination of non-structural and structural proteins. In this study, we demonstrate that immunization with a biologically active HIV-1 Tat protein in combination with the oligomeric HIV-1 gp140 DeltaV2Env and/or SIV Gag proteins, delivered intranasally with the detoxified LTK63 mucosal adjuvant, whose safety has been recently shown in humans, elicits long-lasting local and systemic antibody and cellular immune responses against the co-administered antigens in a fashion similar to immune responses induced by vaccination with Tat, DeltaV2Env and Gag proteins alone. The results indicate lack of antigen interference implying that HIV-1 Tat is an optimal co-antigen for combined vaccine strategies employing DeltaV2Env and/or Gag proteins.

According to recent estimates, 39.5 million people have been infected with HIV and 2.9 million have already died. The effect of HIV infection on individuals and communities is socially and economically devastating. Although antiretroviral drugs have had a dramatically beneficial impact on HIV-infected individuals who have access to treatment, it has had a negligible impact on the global epidemic. Therefore, the need for an efficacious HIV/AIDS vaccine remains the highest priority of the world HIV/AIDS agenda. The generation of a vaccine against HIV/AIDS has turned out to be extremely challenging, as indicated by20 years of unsuccessful attempts. This review discusses the major challenges in the field and key experimental evidence providing a rationale for the use of non-structural HIV proteins, such as Rev, Tat and Nef, either in the native form or expressed by viral vectors such as a replicating adeno-vector. These non-structural proteins alone or in combination with modified structural HIV-1 Env proteins represent a novel strategy for both preventative and therapeutic HIV/AIDS vaccine development.

A plasmid DNA containing two different expression cassettes was prepared to independently drive antigen 85B (85B) of Mycobacterium tuberculosis and HIV-Tat in C57BL/6 mice. In vivo expression of the plasmid was demonstrated by efficient transcription of 85B and Tat mRNAs in mouse fibroblasts. DNA-85B or DNA-(85B-Tat) were immunogenic and protected mice to the same extent against M. tuberculosis infection, with a decrease in the numbers of CFU lung−1 in comparison with nonimmunized animals down to levels (0.64 log10 CFU) not significantly different from protection conferred by bacillus Calmette-Guerin vaccine (0.97 log10 CFU decrease). Multipromoter plasmids, which permit the reduction of the total amount of DNA injected, can be useful for DNA vaccination against tuberculosis.

The need for an effective HIV/AIDS vaccine is imperative to halt a pandemic that involves more than 40 million individuals worldwide as of 2005 and is causing enormous socio-economic losses, especially in developing countries (DC). The overall failure of more than two decades of HIV vaccine research justifies the demands for a concerted effort for the rapid development of new and efficacious vaccines against HIV/AIDS. In this context, building international collaborative networks is a must for speeding up scientific research and optimizing the use of funding in a synergistic fashion, as resources for HIV/AIDS are limited and do not involve most of the biggest Pharmas that are more interested in drug discovery. The AIDS Vaccine Integrated Project (AVIP) consortium is an example of synergistic partnership of international European Union and DC experts with a common research goal. AVIP is a European Commission-funded (FP-6), consortium-based, 5-year program directed to the fast development of new HIV/AIDS vaccine candidates to be tested in phase I clinical trials in Europe for future advancement to phase II/III testing in DC. To ensure their rapid development, AVIP novel combined vaccines include both regulatory and structural HIV antigens, which have already been tested, as single components, in phase I clinical trials. In particular, such combination vaccines may be superior to earlier vaccine candidates, the vast majority of which are based only on either structural or regulatory HIV products. In fact, the generation of immune responses to both types of viral antigens expressed either early (regulatory products) or late (structural products) during the viral life cycle can maximize immune targeting of both primary or chronic viral infection. Further, the rational design of combined vaccines allows exploitation of immunomodulatory functions of HIV regulatory proteins, which can improve immunity against structural vaccine components. The building of the AVIP consortium and its scientific strategy will be reviewed in this paper as an example of the establishment of a consortium regulated by a specific intellectual property agreement.

The Delta-nef live attenuated virus vaccine approach offered in the SIV-macaque model the opportunity to identify humoral and cell-mediated immune responses associated with protection against viral infections. In addition, soluble factors different from those related to specific immune responses appear to correlate with the establishment and maintenance of the protective status.Investigated were: 1) the ability of CD8+ T cells from cynomolgus monkeys vaccinated with SIV Delta-nef and long-term protected against sequential SIVs and SHIV challenges to inhibit in vitro SHIV replication in an acute infection cell system, 2) the ability of cell-free supernatants from CD8+ T cell cultures to inhibit replication of HIV in chronically infected cells, and 3) whether the antiviral activity of CD8+ T cells correlated with IFNgamma production.Soluble factor(s) secreted by CD8+ T cells from Delta-nef vaccinated monkeys significantly inhibited SHIV replication in an autologous cell system. This effect was not dependent on beta-chemokine secretion and correlated with an increased IFNgamma production. In addition, since supernatants from CD8+ T cells inhibited HIV production in chronically infected monocytic cells, the suppressive activity was not related to the viral strain.Vaccination with the live attenuated virus induces both a CD8+ T cell-dependent antiviral activity and IFNgamma responses potentially responsible for the protection from challenge with heterologous highly pathogenic SHIV89.6P. It is conceivable that boosting the "natural" along with the antigen-specific immunity is a desirable outcome to improve the protective efficacy of any vaccine approach.

The promising results obtained with the HIV-1 Tat-based vaccines in mice, monkeys and humans, a better understanding of Tat immunomodulatory functions, as well as evidence that vaccination with trimeric V2 loop-deleted HIV-1 Env induces cross-clade neutralizing antibodies led to the rational design of a novel vaccine based on the combination of Tat and V2-deleted Env.

A major goal of HIV-1 vaccine development is the induction of mucosal immune responses able to stop or reduce viral infection directly at the portal of entry. We established a heterologous prime/boost vaccination protocol based on intradermal priming with the HIV-1 Tat protein and intranasal boosting with the Tat protein co-administered with the mucosal adjuvant MALP-2. Strong Tat-specific humoral responses were elicited in vaccinated mice at both systemic and mucosal levels. The cellular responses were characterized by a Th1 dominant helper pattern. The heterologous prime/boost regimen was also able to induce Tat-specific CTL, which were absent in animals receiving the homologous prime boost scheme. Thus, the heterologous prime/boost protocol was the only regimen able to evoke both CTL and sIgA responses. This suggests that a similar approach can be exploited to develop multi-component vaccines against HIV-1 infections able to induce both systemic and mucosal immune responses.

By the end of 2004, more than 20 HIV-1 vaccine candidates will have entered clinical testing in at least 30 trials worldwide. Almost half of these vaccines include nonstructural HIV-1 gene products. This represents an important innovation in the HIV vaccine field, because until 9 years ago not even preclinical testing in small animal models had been carried out with such immunogens. This review briefly discusses the experimental evidence that provides the rationale for the use of nonstructural HIV-1 gene products as vaccine antigens, and summarizes the current status and the future development of these novel vaccines.

Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.

Evidence is accumulating that CD4 + T-helper (Th) responses play a critical role in facilitating effector responses which are capable of controlling and even preventing human immunodeficiency virus (HIV) infection. The present work was undertaken to determine whether immunization with multiple antigens influenced individual Th responses and increased protection relative to a single antigen. Rhesus macaques were primed with DNA and boosted (immune-stimulating complex-formulated protein) with a combination of regulatory and structural antigens (Tat-Env-Gag) or with Tat alone. Immunization with combined antigens reduced the magnitude of the responses to Tat compared to the single-antigen immunization. Interestingly, the Th immune responses to the individual antigens were noticeably different. To determine whether the qualitative differences in vaccine-induced Th responses correlated with vaccine efficacy, animals were challenged intravenously with simian/human immunodeficiency virus (strain SHIV 89.6p ) 2 months following the final immunization. Animals that developed combined Th1- and Th2-like responses to Gag and Th2 dominant Env-specific responses were protected from disease progression. Interestingly, one animal that was completely protected from infection had the strongest IFN-γ and interleukin-2 (IL-2) responses prior to challenge, in addition to very strong IL-4 responses to Gag and Env. In contrast, animals with only a marked vaccine-induced Tat-specific Th2 response (no IFN-γ) were not protected from infection or disease. These data support the rationale that effective HIV vaccine-induced immunity requires a combination of potent Th1- and Th2-like responses best directed to multiple antigens.

Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1–86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1–20 and 46–60. Furthermore, their splenocytes proliferated and secreted IFN- and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11–40 of Tat. © 2003 Elsevier Science Ltd. All rights reserved.

Two novel classes of biocompatible core-shell anionic microspheres, composed of an inner hard insoluble core, either made of poly(styrene) (PS) or poly(methyl methacrylate) (PMMA), and a soft outer tentacular shell made of long soluble negatively charged arms derived from the steric stabilizer, hemisuccinated poly(vinyl alcohol) or Eudragit L100/55, respectively, were prepared by dispersion polymerization and characterized. Five types of these novel microspheres, two made of poly(styrene) and hemisuccinated poly(vinyl alcohol) (A4 and A7), and three made of poly(methyl methacrylate) and Eudragit L100/55 (1D, 1E, H1D), differing for chemical composition, size, and surface charge density were analyzed for the delivery of the HIV-1 Tat protein for vaccine applications. All microspheres reversibly adsorbed the native biologically active HIV-1 Tat protein preventing Tat from oxidation and maintaining its biological activity, therefore increasing the shelf-life of the Tat protein vaccine. The microspheres efficiently delivered Tat intracellularly, and were not toxic in vitro nor in mice, even after multiple administrations. These results indicate that these novel microparticles are safe and represent a promising delivery system for vaccination with Tat, as well as for other subunit vaccines, particularly when a native protein conformation is required.

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.

The immune response induced by a DNA construct expressing the E2 envelope glycoprotein of bovine viral diarrhoea virus (BVDV) was studied in cattle. Four groups of five calves, were immunised by intradermal injection with a total of 1 mg of plasmid DNA on each of two occasions, with a 3-week dose interval. Group 1 received non-coding plasmid DNA only (control), group 2 received the E2 coding plasmid (0.5 mg) plus non-coding plasmid DNA (0.5 mg) and groups 3 and 4 received the E2 coding plasmid plus plasmid encoding either bovine interleukin 2 (IL-2) or granulocyte macrophage colony stimulating factor (GM-CSF) respectively. Two weeks after the final immunisation, all calves were challenged by intranasal inoculation with 5 ×10 6 TCID50 of homologous virus. On the day of challenge, neutralising antibodies were detectable in 13 of 15 vaccinated calves (one animal in each of groups 3 and 4 remained seronegative at this point). Thereafter, a strong anamnestic serological response was evident in all vaccinated animals. Furthermore, T-cell proliferation following in vitro re-stimulation with BVDV antigen was significantly elevated in the cytokine adjuvanted groups. This enhancement of BVDV specific immune responses in vaccinated animals was reflected in the clinical responses observed post-challenge. In particular, reduced febrile responses provided evidence of a disease sparing effect of vaccination. Significantly, whilst a transient viraemia was detected in all control animals following challenge, no virus was isolated from the leucocytes from 8 out of the 15 vaccinated animals. In groups 2 and 4, three animals remained virus free, although virus was isolated from two animals in each group at a single time point, while in group 3, three out of five animals had detectable viraemia. In summary, the administration of a DNA vaccine encoding only the E2 glycoprotein of BVDV induced a disease sparing effect in vaccinated calves following challenge and protected more than half of the vaccinated animals from detectable viraemia. © 2002 Elsevier Science Ltd. All rights reserved.

Cytotoxic T cell responses are key to the control of intracellular pathogens including HIV-1. In particular, HIV-1 vaccines based on regulatory proteins, such as Tat, are aimed at controlling HIV-1 replication and at blocking disease development by inducing cytotoxic T cell responses. Naked DNA is capable of inducing such responses but it requires several inoculations of high amounts of DNA, and/or prime-boost regimens. Here, we show that a novel class of cationic block copolymers protect the DNA from DNAse I digestion, and improve DNA delivery to antigen-presenting cells (APCs) after intramuscular (i.m.) vaccination. In particular, three cationic block copolymers (K1, K2 and K5) were used to deliver the HIV-1 pCV-tat DNA vaccine in BALB/c mice. The results indicate that vaccination with a very low dose (1 microg) of pCV-tat delivered by the cationic block copolymer K2 is safe and, as compared to naked DNA (up to 30 microg), greatly increases the CTL response against Tat, which was detected in all animals in the absence or in the presence of re-stimulation.

Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the tat gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.

Recent evidence suggests that a CD8-mediated cytotoxic T cell response against the Tat protein of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) controls primary infection after pathogenic virus challenge, and correlates with the status of long-term nonprogressor in humans. Due to the presence of unmethylated CpG sequences, DNA vaccination can boost the innate immunity driving more potent T cell-mediated immune responses. Therefore, cynomolgus monkeys were vaccinated with a tat-expressing vector containing defined unmethylated CpG sequences (pCV-tat). Here it is shown that the intramuscular inoculation of the pCV-tat contained primary infection with the highly pathogenic SHIV89.6P virus preventing the CD4(+) T cell decline in all the vaccinated monkeys. Undetectable virus replication and negative virus isolation correlated in all cases with the presence of anti-Tat CTLs. However, a CD8-mediated non cytolytic antiviral activity was also present in all protected animals. Of note, this activity was absent in the controls but was present in the monkey inoculated with the CpG-rich vector alone that was partially protected against viral challenge (i.e. no virus replication but positive virus isolation). These results suggest that a CTL response against Tat protects against primary infection by blocking virus replication at its early stage, in the absence of sterilizing immunity. Nevertheless, the boost of the innate immunity by CpG sequences can contribute to this protection both by driving more potent CTL responses and by inducing other CD8-mediated antiviral activities. Thus, the CpG-rich tat DNA vaccine may represent a promising candidate for preventive and therapeutic vaccination against AIDS.

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi’s sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-α), we analyzed the effects of IFN-α or anti-IFN-α antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-α inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-α inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-α was also observed in BC-1 cells induced with n -butyrate. Conversely, the addition of anti-IFN-α Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-α Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-α reduced the HHV-8 load in PBMC. Thus, IFN-α appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-α may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.