Roberto Belli

Control of SHIV-89.6P-infection of cynomolgus monkeys by HIV-1 Tat protein vaccine

Vaccine strategies aimed at blocking virus entry have so far failed to induce protection against heterologous viruses. Thus, the control of viral infection and the block of disease onset may represent a more achievable goal of human immunodeficiency virus (HIV) vaccine strategies. Here we show that vaccination of cynomolgus monkeys with a biologically active HIV-1 Tat protein is safe, elicits a broad (humoral and cellular) specific immune response and reduces infection with the highly pathogenic simian-human immunodeficiency virus (SHIV)-89.6P to undetectable levels, preventing the CD4+ T-cell decrease. These results may provide new opportunities for the development of a vaccine against AIDS.

Vaccination with DNA containing tat coding sequences and unmethylated CpG motifs protects cynomolgus monkeys upon infection with simian/human immunodeficiency virus (SHIV89.6P)

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.

SHIV89.6P pathogenicity in cynomolgus monkeys and control of viral replication and disease onset by human immunodeficiency virus type 1 Tat vaccine

The Tat protein of human immunodeficiency virus (HIV) is produced very early after infection, plays a key role in the virus life cycle and in acquired immunodeficiency syndrome (AIDS) pathogenesis, is immunogenic and well conserved among all virus clades. Notably, a Tat‐specific immune response correlates with non‐progression to AIDS. Here, we show that a vaccine based on the Tat protein of HIV blocks primary infection with the simian/human immunodeficiency virus (SHIV)89.6P and prevents the CD4 T cell decline and disease onset in cynomolgus monkeys. No signs of virus replication were found in five out of seven vaccinated macaques for almost 1 year of follow‐up. Since the inoculated virus (derived from rhesus or from cynomolgus macaques) is shown to be highly pathogenic in cynomolgus macaques, the results indicate efficacy of Tat vaccination in protection against highly pathogenic virus challenge. Finally, the studies of the Tat‐specific immunological responses indicate a correlation of protection with a cytotoxic T cell response. Thus, a Tat‐based vaccine is a promising candidate for preventive and therapeutic vaccination in humans.

Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys.

The ability of a live attenuated simian immunodeficiency virus (SIV) to protect against challenge with cloned SIVmac251/BK28 was evaluated in four cynomolgus macaques. The intravenous infection of the C8 variant of the SIVmac251/32H virus, carrying an in-frame 12 bp deletion in the nef gene, did not affect the CD4+ and CD8+ cell counts, and a persistent infection associated with an extremely low virus burden in peripheral blood mononuclear cells (PBMCs) was established. After 40 weeks, these monkeys were challenged intravenously with a 50 MID50 dose of SIVmac251/BK28 virus grown on macaque cells. Four naive monkeys were infected as controls. Monkeys were monitored for 62 weeks following challenge. Attempts to rescue virus from either PBMCs or bone marrow from the C8-vaccinated monkeys were unsuccessful, but in two cases virus was re-isolated from lymph node cells. The presence of the SIV provirus with the C8 variant genotype maintaining its original nef deletion was shown by differential PCR in PBMCs, lymph nodes and bone marrow. Furthermore, in contrast to the control monkeys, the vaccinated monkeys showed normal levels for CD4+ and CD8+ cells, minimal lymphoid hyperplasia and no clinical signs of infection. Our results confirm that vaccination with live attenuated virus can confer protection. This appears to be dependent on the ability of the C8 variant to establish a persistent but attenuated infection which is necessary for inducing an immune response, as suggested by the persistence of a strong immune B cell memory and by the over-expression of interleukin (IL)-2, interferon-gamma and IL-15 mRNAs in PBMCs of C8-vaccinated monkeys but not in those of control monkeys.

A combination HIV vaccine based on Tat and Env proteins was immunogenic and protected macaques from mucosal SHIV challenge in a pilot study

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.

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.

T-cell-mediated protective efficacy of a systemic vaccine approach in cynomolgus monkeys after SIV mucosal challenge.

Abstract:  The immunogenicity and the protective efficacy of a new polyvalent triple vector (DNA/SFV/MVA) based vaccine against mucosal challenge with pathogenic SIVmac251 were investigated. Cynomolgus monkeys (Macaca fascicularis) were primed intradermally with DNA, boosted twice subcutaneously with recombinant Semliki Forest virus (rSFV) and finally intramuscularly with recombinant Modified Vaccinia Virus Ankara strain (rMVA). Both DNA and recombinant viral vectors expressed SIV proteins (Gag, Pol, Tat, Rev, Nef and Env). The vaccinated monkeys developed T helper proliferative responses to viral antigens after the second immunization while interferon (IFN)‐γ enzyme‐linked immunosorbent spot‐forming cell assay (ELISPOT) specific responses appeared only after the last boost with rMVA. Upon intrarectal challenge with pathogenic SIVmac251, three of four vaccinated monkeys were either fully protected or exhibited a dramatic reduction of virus replication up to undetectable level. A major contribution to this protective effect appeared to be the anamnestic T‐cell IFN‐γ ELISPOT responses to vaccine antigens (Gag, Rev, Tat, Nef) that mirrored the viral clearance. These results underline the efficacy of a multiprotein approach in combination with a triple vector system of antigen delivery.

Containment of Infection in Tat Vaccinated Monkeys After Rechallenge with a Higher Dose of SHIV89.6Pcy243

We previously reported that cynomolgus monkeys vaccinated with the human immunodeficiency virus (HIV)-1 Tat protein controlled infection after challenge with the simian human immunodeficiency virus (SHIV) 89.6P(cy243) for up to 2 y of follow-up. To evaluate the breadth of the protective immunity elicited by the Tat protein, the vaccines along with the naïve monkeys were intravenously rechallenged with a fivefold higher dose (50 MID(50)) of the same SHIV-89.6P(cy243). The vaccinated monkeys exhibited a statistically significant and long-lasting reduction of viral replication compared to control monkeys. This effect was associated with a strong anamnestic response to Tat, while responses to Gag and Env were nearly undetectable. Taken together, these data provide further evidence for the usefulness of Tat-based vaccines.

Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIVmacJ5 on the kinetics of SIV replication in cynomolgus monkeys

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIVmac251/32H/J5) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA‐J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIVmac251. All control monkeys, previously inoculated with non‐recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post‐challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA‐J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell‐ and plasma‐associated viral load.

Influence of MHC class I and II haplotypes on the experimental infection of Mauritian cynomolgus macaques with SHIVSF162P4cy

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.