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

Abstract

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 MID₅₀ (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.

Introduction

Currently, HIV/AIDS vaccine candidates based on the combination of structural and regulatory HIV proteins may hold the best promise for developing a protective vaccine [1]. In fact, the overall disappointing results of HIV/AIDS vaccine phase IIb/III clinical trials conducted to date [2], [3] can be, at least in part, ascribed to the inadequacy of classical vaccine strategies based on HIV structural antigens (Env, and/or Gag, and Pol), which failed to provide protection [4]. On the other hand, less traditional vaccine approaches with HIV regulatory proteins (Tat, Rev, and Nef) were able to contain virus replication and preventing disease onset and progression in preclinical studies [1], [4]. More recently, novel vaccine modalities encompassed both structural and regulatory HIV proteins to generate antibody (Ab) and cell-mediated immunity against multiple HIV components that are key for HIV entry/replication. Such combined vaccines are intended to block/control early and late HIV infection-related events, to contain acute virus infection and/or protect the host from disease progression [1], [4], [5]. The exploitation of HIV regulatory proteins’ immunomodulatory properties [6], [7] represents an added value of this combined vaccine approach [1].

We describe here the rational design and evaluation in macaques of an innovative HIV/AIDS vaccine based on the combination of HIV Tat and Env proteins, aimed at inducing protective immunity capable of neutralizing the virus as well as Tat biological activities [8], [9], [10], [11]. In particular, this study was conceived as a pilot study to evaluate the effects of native Tat protein addition to an Env-based vaccine. To this aim, the two pivotal experimental groups consisted of macaques immunized with Env only or with Tat and Env. Control groups included animals inoculated with the adjuvants only or left untreated (naïve), and one animal vaccinated with the native Tat protein alone.

An oligomeric, SF162 strain-derived, V2 loop-deleted Env (EnvΔV2) was chosen for its capacity of eliciting neutralizing antibodies (nAbs) against primary HIV isolates [12]. The deletion of the V2 loop allows exposure of conserved neutralization-sensitive epitopes to increase the breadth of vaccine-elicited Ab responses, thus potentially circumventing the issue of HIV Env intra- and inter-clade variability [13].

The inclusion of biologically active, HIIIB strain-derived Tat protein in the vaccine regimen was intended to generate immune responses against the early viral product Tat and to exploit Tat immunomodulatory properties.

Tat is key for virus replication, cell-to-cell virus transmission, and HIV pathogenicity, and can enter both infected and uninfected cells, modulating the expression of cellular genes [14]. Biologically active Tat promotes monocyte-derived dendritic cells maturation towards a Th-1 polarizing phenotype as well as their antigen-presenting activity, leading to a more efficient presentation of both allogeneic and exogenous soluble antigens, in vitro [15], [16]. By modulating the composition and activity of the immunoproteasome, Tat changes the hierarchy of CTL epitopes in favor of subdominant and cryptic ones and shows vaccine-adjuvant properties [17], [18], [19], [20].

Tat is highly conserved in its immunodominant domains representing, in principle, an ideal target for a broadly effective vaccine [21]. Because of its multiple functions, immune responses against biologically active Tat can contribute to the control of HIV infection and/or disease progression. Of note, anti-Tat cellular or humoral immunity was reported to correlate, in the course of natural infection, with early virus control [22], [23] or with asymptomatic infection and long-term non-progression to AIDS [24], [25], respectively. Furthermore, a retrospective analysis conducted on 112 cynomolgus macaques indicated that vaccination with the biologically active Tat protein reduced the rate of infection acquisition against challenge with 10 MID₅₀ of pathogenic SHIV89.6P, contained acute CD4⁺ T cell depletion against 15 MID₅₀ of virus, and contained CD4⁺ T cell loss in the chronic phase of infection, regardless of the challenge dose [26]. Recently, the HIV Tat vaccine has shown to be safe and immunogenic in preventive and therapeutic phase I trials [27], [28] and to intensify the therapeutic effects of HAART in successfully treated HIV-infected individuals, making Tat a promising target of an HIV/AIDS vaccine [29].

The experimental design of this pilot study consisted of multiple subcutaneous and intranasal immunizations with proteins in adjuvants, followed by assessment of immune responses at both the systemic and the mucosal levels. The Alum adjuvant (aluminum phosphate), which is commonly used in licensed human vaccines [30], was employed for subcutaneous immunizations, whereas adjuvant LT-K63 was given intranasally [31]. LT-K63 is an Escherichia coli heat-labile enterotoxin mutant, which proved generally safe and effective as an intranasal adjuvant both in animals and in humans [31], [32], [33]. In particular, multiple LT-K63 intranasal administrations in animals did not induce histological inflammatory changes in the respiratory tract or olfactory bulbs and in the meninges [31]. When co-administered with a large number of immunogens by different routes in mice, LT-K63 was able to enhance immune responses [31]. In female rhesus macaques immunized with the HIV Env protein and LT-K63 adjuvant either intramuscularly, or intramuscularly and intranasally, protection was achieved against intravaginal simian-human immunodeficiency virus (SHIV) challenge [33]. LT-K63 also showed an overall good safety profile and intranasal adjuvanticity for influenza in humans [32]. Nonetheless, more recently (after the completion of our monkey study), two phase I clinical trials [34] on nasal subunit vaccines against HIV and tuberculosis employing LT-K63 as an adjuvant confirmed previous concerns on the association of LT-K63-containing nasal influenza vaccine and Bell's palsy [35].

Efficacy of the Tat/EnvΔV2 or EnvΔV2 alone vaccines was evaluated against homologous intrarectal challenge with an R5 SHIV.