NATAP: Topical DermaVir May Control HIV in Monkeys

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Control of viral rebound through therapeutic immunization with DermaVir (in monkeys)

AIDS: Volume 19(1) 3 January 2005

Lisziewicz, nnaa,b; Trocio, a; Xu, Jianqinga; Whitman, Luciaa; Ryder, Amya; Bakare, Nyashaa; , Mark Gc; Wagner, Wendelinec; Pistorio, d; Arya, Sureshe; Lori, Francoa

aResearch Institute for Genetic and Human Therapy (RIGHT)

bGenetic Immunity, LLC, Washington DC

cSouthern Research Institute, Frederick, land, USA

dClinical Epidemiology and Biometry Unit, IRCCS G. Gaslini, Genova, Italy

eNational Institutes of Health, National Cancer Institute, Bethesda, land, USA

The authors say:

--Experimental evidence described here demonstrated that DermaVir immunization provided virological, immunological and clinical benefit for chronically infected macaques

-- following DermaVir-HAART treatment cycle, the median viral load rebound during antiretroviral treatment interruption decreased ….subsequent cycles of DermaVir-HAART further decreased viral load rebound to a median of 460 and then to <200 copies/ml

-- As DermaVir immunization was more effective in combination with antiretroviral drugs we do not view DermaVir as a substitute for antiretroviral drugs, rather as a potential new therapy with a unique mechanism of action

-- Here we have demonstrated that the repeated immunization schedule is feasible and desirable treatment approach with DermaVir. The topical administration and absence of common toxicities with antiretroviral drugs might be also appealing to patients.

-- DermaVir utilizes a needle-free, topical application that can be manufactured for large-scale human use and repeatedly administered

FOR KEY DETAILS OF STUDY REPORT READ BELOW THE STUDY RESULTS & AUTHOR DISCUSSION

ABSTRACT

Objective: To reconstitute immune responses capable of eliminating infected cells and suppressing viral load during chronic retroviral infection.

Design: A topical, DNA-based therapeutic immunization (DermaVir) was designed to express most of the regulatory and structural viral genes in dendritic cells.

Methods: DermaVir alone and in combination with antiretroviral drugs was tested in chronically SIV-infected macaques.

Results: DermaVir provided virological, immunological and clinical benefit for SIV-infected macaques during chronic infection and AIDS. In combination with antiretroviral drugs, DermaVir augmented SIV-specific T-cell responses and enhanced control of viral load rebound during treatment interruptions.

Conclusions: The results indicate the feasibility of therapeutic immunization even in immune compromised hosts, and suggest that DermaVir can complement antiretroviral drugs to sustain suppression of HIV-1 replication.

INTRODUCTION

The treatment of HIV/AIDS involves combination of antiretroviral drugs targeting various stages of the virus life cycle, which enables suppression of viral replication over months to years. Clinical experience has demonstrated that prolonged exposure to antiretroviral drugs is associated with significant adverse effects and increasing emergence of drug resistant mutations that limit the durability of HIV-1 suppression. Even a short interruption of therapy leads to viral load rebound in most individuals [1], because HIV-1-specific immune responses are not reconstituted, but decline with time on treatment [2]. Since virus-specific immunity can control viral replication [3], one approach to achieve durable suppression of viremia is to enhance the HIV-specific T-cell responses in infected individuals.

To boost immunity and provide a new treatment approach for HIV-1-infected individuals complementing the existing therapies we have been developing DermaVir immunization. DermaVir is a novel topical vaccine designed to improve antigen presentation and induce cytotoxic T-cell responses for the treatment of HIV/AIDS. DermaVir delivers plasmid DNA authentically expressing most of the structural and regulatory genes of the virus into epidermal Langerhans' cells. Langerhans' cells mature to dendritic cells and migrate to the draining lymph nodes. DermaVir-derived gene expression occurs in dendritic cells capable of converting naive T-cells to functional cytotoxic T-cells and polarizing the immune system towards T-cell-mediated immune responses [4]. Since the immune system of HIV-infected individuals has already been primed by large amounts of viral antigen, the purpose of DermaVir therapy is to improve the presentation of viral antigens in order to induce functional T-cell responses that can control viral replication.

Here we provide evidence that therapeutic immunization with DermaVir progressively contains viral load rebound during chronic SIV infection and AIDS.

RESULTS

We first examined a cohort of 10 late-stage, chronically SIVmac251-infected rhesus macaques with AIDS. Disease progression in SIV-infected monkeys is faster than in HIV-1 infected humans, and AIDS occurs generally within 1 year of infection, at which time around 70% of the animals die. In fact, during the protocol approval period, three animals died. Fourteen months after infection, we randomized the remaining seven animals such that three animals received a continuous highly active antiretroviral therapy (HAART) and four animals received the same HAART regimen administered intermittently (3 weeks on/3 weeks off therapy) [5].

Continuous HAART successfully suppressed virus replication and increased CD4 T-cell counts. After 6 months of treatment, animals experienced viral breakthrough (presumably because of the onset of drug resistance) and CD4 T-cell loss, and died. Intermittent antiretroviral therapy also suppressed virus replication and recovered CD4 T-cell counts during HAART cycles, but did not suppress viral load rebound during six consecutive interruption cycles. After six therapy cycles, the median viral load rebound had increased to 4 292 260 copies/ml, a sign of imminent risk of death in this infection (one animal did die during treatment). The lack of induction of suppression of virus replication by intermittent HAART cycles in these animals, in contrast to macaques treated early after infection [5], simulated what has been observed in humans [1,6].

We amended the protocol to administer multiple doses of DermaVirSHIV in combination with HAART. Following the first DermaVir-HAART treatment cycle, the median viral load rebound during antiretroviral treatment interruption decreased from 4,292,260 to 12,000 copies/ml. Subsequent cycles of DermaVir-HAART further decreased viral load rebound to a median of 460 and then to <200 copies/ml. Rate of viral load rebound decreased from 0.26 to 0.09, 0.01, and 0 log10/day, similar to what was observed in macaques treated with intermittent administration of HAART during acute infection [5]. Although all of the animals responded consistently to DermaVirSHIV treatment, their individual profiles were slightly different. The response of macaque #51 mirrored the median responses. Macaque #56 responded faster, and viral load rebound was completely suppressed after two cycles of DermaVir-HAART. Macaque #60, which was partially unresponsive to HAART prior to DermaVirSHIV treatment, did not respond to DermaVir-HAART with complete viral suppression, nevertheless his viral load also progressively decreased.

There was an association between viral load inhibition and induction of SIV-specific T cells expressing interferon (IFN)-γ, as quantified by flow cytometry. Since these macaques had AIDS-associated hematological disorders, we could only obtain adequate specimens from two of them, #51 and #56. In both macaques, SIV-specific T-cell responses progressively increased after every cycle of DermaVir-HAART.

To expand and confirm our findings, we conducted a randomized, controlled non-human primate trial. In this study macaques were also infected with SIVmac251, but treatment was started 6 months after infection, corresponding in this model to an established chronic asymptomatic infection in humans, that is a stage of infection representing the majority of HIV-1-infected patients. We also wanted to explore whether DermaVir immunization in the presence or in the absence of therapy would provide comparable results. We randomized macaques based on viral load to four groups: untreated, treated with DermaVirSHIV, intermittent HAART, or DermaVirSHIV plus intermittent HAART for 42 weeks.

The median viral load of the control group differed from all experimental groups significantly (Dunnet test, P < 0.0001), including the group treated with DermaVirSHIV alone. In fact, some of the animals responded to DermaVirSHIV and transiently lowered viremia after immunizations, a direct evidence of DermaVir antiretroviral efficacy. CD4 T-cell counts of the control animals, higher than the DermaVirSHIV group at baseline (Wilcoxon signed rank test, P = 0.066), decreased to a greater extent than those of the animals treated with DermaVirSHIV. The disease in the untreated control animals progressed faster than in the other groups, and at week 42 five of six macaques in the control group had died as compared to three of six macaques treated with DermaVirSHIV alone.

In the intermittent HAART group, viral load significantly rebounded (Mann-Whitney U test, P < 0.0001) during the treatment interruption cycles to a median level that remained constant throughout the course of follow up (Fig. 3c), except for one macaque in this group (16%) that controlled SIV replication, similar to the frequency of control of HIV-1 replication (17%) documented in chronically infected patients treated with intermittent HAART [1]. In contrast, intermittent treatment with DermaVir-HAART induced a progressive containment of viral load rebound during treatment interruption cycles in the majority of the animals. Consistent with the previous study, the median viral load during treatment interruption became undetectable (<200 copies/ml) in the DermaVir-HAART group after the first two cycles, and it remained undetectable with the exception of a modest rebound at week 36 (700 copies/ml). The difference between the DermaVir-HAART and intermittent HAART groups during the on-therapy observation points was not significant, (Mann-Whitney U test, P = 0.14), as antiretroviral drugs effectively suppressed SIV in both groups; however, differences were significant between the two groups when comparing the off-therapy peaks of viremia (Mann-Whitney U test, P = 0.0017).

CD4 T-cell counts fluctuated in both intermittent HAART and DermaVir-HAART treated groups and typically increased during treatment and decreased during treatment interruption cycles (Fig. 3d). CD4 count and percentage changes between baseline and week 42 did not significantly differ among groups (Mann-Whitney U test, P = 0.24 and P = 0.31, respectively). The use of hydroxyurea, a cytostatic drug known to blunt the CD4 increase in HIV infected patients [7], might explain the lack of CD4 increase despite virologic control in the DermaVir-HAART group.

Animals were followed for 60 weeks post-infection. Chronically infected monkeys treated with DermaVir alone, DermaVir-HAART and intermittent HAART survived longer than the untreated control group (Fig. 3e).

According to the ICC assay, SIV-specific T-cells were negligible prior to initiation of antiretroviral therapy and remained low both in the control macaques and in the macaques treated with DermaVirSHIV alone (not shown). By the fifth and sixth treatment cycle, intermittent HAART had increased the frequency of SIV-specific CD8 T cells and CD4 T-helper cells during both treatment and treatment interruption cycles. The expansion of SIV-specific CD8, but not CD4 T cells, was more pronounced in the DermaVir-HAART treatment group.

The ELISPOT analysis with SIV peptides confirmed and expanded the ICC results (Fig. 4c). We found increased T-cell frequencies in the DermaVir-HAART group compared to intermittent HAART group. With respect to the breadth of response, repeated DermaVirSHIV administration expanded SIV-specific Gag and Nef immune responses, but did not influence SIV-specific Tat and Rev responses. This finding is consistent with the fact that the SHIV-derived DNA construct in DermaVirSHIV expresses SIV Gag and Nef and HIV-1 Tat and Rev proteins and suggests the DNA specificity of the DermaVir vaccine treatment.

The main side effect of DermaVir immunization observed in the macaque studies was a mild, reversible local skin irritation. To confirm the findings, a GLP toxicology study was performed in the swine model, an appropriate one for determining local reactogenicity and systemic toxicity of topical vaccines, because porcine skin is similar in structure to human skin, particularly with respect to the epidermis. The swine study indicated mild and transient local site reactogenicity associated with skin preparation, lack of apparent systemic toxicity, no differences in safety between male or female animals and lack of persistence of plasmid DNA except in skin tissues, confirming the safety of the product and suggesting that plasmid DNA does not persist after immunization.

AUTHOR DISCUSSION

Experimental evidence described here demonstrated that DermaVir immunization provided virological, immunological and clinical benefit for chronically infected macaques. Viral load suppression by DermaVir was independent of disease stage, and both SIV-specific T-cell responses and control of viral rebound were enhanced by the combination of DermaVir with antiretroviral drugs. The antiviral and immunological activity of DermaVir reveals a previously unexpected capacity of an immune compromised host to respond to immunization and provides optimism that T cell-mediated virus suppression might still be achievable long after initial retroviral infection. It was also encouraging to see that DermaVirSHIV elicited an immune response inhibiting a challenge virus with a different envelope (HIV and SIV respectively). Pre-clinical studies provided relevant data suggesting that DermaVir is unlikely to cause serious adverse reactions in human subjects.

As DermaVir immunization was more effective in combination with antiretroviral drugs we do not view DermaVir as a substitute for antiretroviral drugs, rather as a potential new therapy with a unique mechanism of action. In fact, viral load suppression by T cell-mediated cytotoxicity might be the result of elimination of infected cells, as opposed to viral life cycle interference mediating the efficacy of current antiretroviral drugs. Therapeutic immunizations are expected to have a resistance profile [8] different from those of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and other drugs. As such, therapeutic immunizations might improve the durability of the presently used drugs by preventing viral rebound in case of lack of adherence or when patients temporarily stop drugs due to toxicity [9]. Here we have demonstrated that the repeated immunization schedule is feasible and desirable treatment approach with DermaVir. The topical administration and absence of common toxicities with antiretroviral drugs might be also appealing to patients.

DermaVir represents an advancement in the field of therapeutic immunization [10], as it exerts antiviral activity during chronic and late stage retroviral infection. Others have demonstrated inhibition of SIV replication [11] with dendritic cell-based ex vivo therapeutic immunization early after infection, when the immune system is still preserved and has the best chances of achieving immune reconstitution even with the host's own virus [5,6]. Unfortunately, ex vivo techniques are cumbersome, expensive, and limited to highly specialized laboratories. In contrast, DermaVir utilizes a needle-free, topical application that can be manufactured for large-scale human use and repeatedly administered. The results presented here further support the feasibility of DermaVir immunization for the treatment of chronic HIV infection.

Methods

Composition of DermaVirSHIV and study design

DermaVirSHIV is composed of a mixture of polyethylenimine mannose (PEIm) and plasmid DNA [4], similar to DermaVir for clinical use, but instead of an HIV-based plasmid it contains a SHIV-based plasmid, encoding for most of the regulatory and structural genes except the integrase. Indian rhesus macaques were treated with antiretroviral drugs as described previously [5]. Some animals were also treated topically with DermaVirSHIV every 6 weeks in combination with antiretroviral drugs, twice one week apart, that is 3 and 10 days before interrupting antiretroviral treatment. Two axillary and two inguinal skin sites were shaved, lightly rubbed with an exfoliating sponge, and 0.2 ml DermaVirSHIV solution (0.1 mg DNA) was applied to each of four prepared skin sites. After a treatment time of approximately 40 min under general anesthesia, the animals were returned to their cages. All non-human primate studies described here were performed with protocols approved by the Internal

Animal Care and Use Committee.

Immunologic assays

We performed the intracellular cytokine assay (ICC) as described previously [5]. The ELISPOT assay was performed using a commercially available kit (BD Biosciences). Complete peptide sets were obtained from the NIH AIDS Research and Reference Reagent Program, and combined to form large pools (0.5 mg/ml) covering each of the following genes: SIVmac239 Gag (#6204), SIVmac239 Nef (#6206), SIVmac239 Tat (#6207), and SIVmac239 Rev (#6448).

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