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Vaccination with MV1-F4 induces strong F4- and MV-specific T cell responses in mice

The immunogenicity of MV1-F4 recombinant vaccine was first evaluated in genetically modified CD46-IFNAR mice susceptible to MV infection. Intracellular cytokine staining was detected by flow cytometry following in vitro stimulation of freshly extracted splenocytes with HIV-1 F4 peptide pools (Figure 1A and B) and empty MV (Figure 1C and D). Intracellular cytokine staining for IFNγ and IL-2 was observed in both CD4+ and CD8+ T cells from immunised animals, as compared with non-immunised control mice. The intensity of response, expressed as the percentage of single or double cytokine-positive CD4+ and CD8+ cells, was dependent on the inoculated dose with a marked increase with the highest dose (107 TCID50), resulting in strong HIV and MV responses. Single and double cytokine staining for IFNγ and IL-2 was observed in both HIV F4- and MV-specific CD4+ and CD8+ T cells. However, IFNγ was produced in a much higher amount than IL-2. The percentages of CD4+ T cell cytokine responses were at least 2 times higher than CD8+, both for HIV and MV. Altogether, this analysis shows that MV1-F4 is strongly immunogenic and elicits a high level of CD4+ and CD8+ T cell responses in CD46-IFNAR mice, supporting its further evaluation in non-human primates.

Vaccination with MV1-F4 induces polyfunctional T cell responses to HIV in macaques

Polyfunctional CD4+ and CD8+ T cell cytokine responses were detected by flow cytometry following in vitro stimulation of PBMC with HIV-1 F4 peptide pools (Figure 2). Single, dual and triple cytokine staining for TNFα, IL-2 and IFNγ by CD4+ and CD8+ T cells was observed (Figure 2A–D). The activation marker CD154, also known as CD40 ligand, was co-expressed by cytokine positive CD4+ T cells (Figure 2A and B).

Following vaccination, potent CD4+ T cell cytokine responses against HIV-1 F4 insert peptide pools were detected in macaques F52 and F53 from group A and macaques F56 and F57 from group B (Figure 3A and B). By contrast, macaques F51 and F54 from group A and F55 and F58 from group B did not demonstrate significant CD4+ T cell cytokine responses (Figure 3A and B). The CD4+ T cell response of macaque F55 was ambiguous as it was low and occurred only at a single, late time point (Figure 3B). Only one animal in group A, F52, showed any sign of a boosted CD4+ and CD8+ T cell response against F4 peptides after a second vaccination. The CD4 responses of F53 were already high and continuing to rise at the time of boosting peaking on day 42. Responses in both groups declined sharply at day 84 and could not be detected in lymphoid tissues taken at termination (Figure 3A and B). Significant CD8+ T cell cytokine responses against HIV-1 F4 insert peptide pools were detected in all animals (Figure 3C and D). However, compared with the CD4+ T cell responses against HIV-1 F4 peptides, the magnitude of CD8+ T cell responses was moderate. CD8+ T cell cytokine responses were overall greater and appeared earlier in group B than in group A, but peripheral responses in both groups declined sharply at day 84. Nonetheless, significant HIV-1 F4-specific CD8+ T cell responses could still be detected in spleen cells collected over 3 months after the last immunisation, from all macaques except F51 and F53 (Figure 3C and D).

Vaccination with MV1-F4 induces humoral responses to HIV in macaques

Macaques F51, F52, F53, F54 and F57 developed binding antibodies against HIV-1 F4 antigen as soon as 14 days after immunisation with MV1-F4 (Figure 5A). A very low F4-specific binding antibody response was detected in the F55 animal only at day 14. Seroconversion of macaque F51 was only detected after a second immunisation (Figure 5A). Titres of anti-F4 binding antibodies in macaques F52, F53 and F54 were boosted after a second immunisation, while the anti-F4 humoral responses in the F57 animal waned at day 56. Macaques F56 and F58 did not seroconvert to the F4 antigen (Figure 5A).

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Several therapeutic approaches are being considered to control or eliminate the HIV latent reservoir. These involve either a complete elimination of all persistent HIV (sterilizing cure) or the immunological control of persistent HIV (functional cure). The “shock and kill” approach is the main focus of current research efforts for a sterilizing cure. In this approach, small molecules that activate HIV transcription would be used to force the reactivation of latent HIV in memory CD4+ T cells under the cover of ART. Subsequently, reactivation of HIV expression would induce viral cytopathic effects, immune clearance, and cell death, thereby purging latently infected cells while uninfected cells are protected by ART (4). Challenges to this approach involve the heterogeneity of latent HIV and the lack of evidence that lymphocytes, in which HIV is reactivated, are eliminated from the latent pool (reviewed in 5–7). Recent studies have also illustrated that different latent viruses might become differentially reactivated in response to different drugs (8–10), and that HIV-latency reactivation might behave in a stochastic rather than deterministic manner when challenged by small molecules (10). Additionally, many of the non-reactivated proviruses appear to be replication competent, indicating that the latent reservoir may be up to 60-fold larger than previously estimated (10).

The complex mechanisms of HIV transcriptional regulation, as well as HIV latency, have been recently summarized in a number of excellent reviews (5–7, 11). In this review, we highlight the core principles that regulate HIV transcription and then focus on two broad mechanisms that control HIV latency: (a) cis-acting mechanisms, dependent on the site of integration of the virus into the genome and the local chromatin environment at that site, and (b) trans-acting mechanisms, including basal and activated transcription factors, their regulation by the state of activation of T cells, and the environmental cues that these cells receive.

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The detection of an increasing number of circulating recombinant strains of human immunodeficiency virus type 1 (HIV-1) indicates that genetic recombination can occur in cells infected with two strains of HIV-1.1,2 Coinfection with two circulating strains of HIV-1 has been detected in a few subjects in communities where HIV-1 infection is endemic.3 Coinfection may result from exposure to a second virus either shortly after the initial infection or during the course of established HIV-1 infection; the latter circumstance is called superinfection.
Most viral infections induce lifelong immunity, but reinfection with respiratory viruses such as respiratory syncytial virus is common, most likely because immunity becomes nonprotective or fades. It is thought that HIV-1 superinfection is a rare event1–6 and that it is prevented by previous viral exposure through a phenomenon called superinfection immunity.5 However, HIV-1 superinfection has been induced experimentally in chimpanzees.7 In this animal model and in superinfection induced with the simian immunodeficiency virus in macaques, the second infection produces a slower deterioration in immunity than does the initial infection,7–9 and there is more efficient control of viremia. In this article, we report a case of HIV-1 superinfection.
Case Report
In November 1998, a 38-year-old man presented with an acute retroviral syndrome. Anti–HIV-1 antibodies were undetectable, the level of p24 antigen was greater than 100 pg per millimeter, the plasma level of HIV-1 RNA was 805,000 copies per milliliter, and the CD4 cell count was 684 per cubic millimeter. Sequences of the HIV-1 reverse transcriptase and protease genes revealed no mutations associated with drug resistance and identified the HIV-1 as subtype AE. For years, the patient had had sexual contacts with multiple unknown male partners. He enrolled in the QUEST trial10 in November 1998 and received highly active antiretroviral treatment (HAART) with zidovudine, lamivudine, abacavir, and amprenavir for 27 months. From month 21 to month 27 he participated in a vaccination trial and was randomly assigned to receive ALVAC vector vCP1452.11
Six weeks after the initiation of HAART, the plasma level of HIV-1 RNA declined to 1000 copies per milliliter. Treatment was then interrupted for six weeks because of toxic effects on the liver. After HAART was resumed, the HIV-1 RNA level decreased rapidly, to less than 50 copies per milliliter (Figure 1). After vaccination, HAART was again interrupted (on January 21, 2001) as part of the vaccine research protocol. In February 2001, the patient's plasma HIV-1 RNA level rose to 80,000 copies per milliliter (the first rebound) and then decreased to 21,000 copies per milliliter. A rapid increase in the HIV-1 RNA level was next observed on April 10 (the second rebound), and for the next four months the level fluctuated between 200,000 and 400,000 copies per milliliter. The patient's symptoms (transient fatigue and fever) were mild, and he declined to resume HAART during this period. His history revealed that he had had several unprotected sexual contacts in Brazil three weeks before the second rebound of viremia. Four months after the second rebound, however, HAART was resumed, and the plasma level of HIV-1 RNA rapidly decreased. Treatment was again interrupted after an increase in the alanine aminotransferase level to 800 U per liter, as compared with a level of 200 U per liter before HAART. At this time, serologic data and quantification of hepatitis C virus (HCV) RNA (Figure 1) documented an acute HCV infection. The increase in alanine aminotransferase was attributed both to HCV infection and to drug-induced toxic effects. Treatment with pegylated interferon and ribavirin was followed by clearance of HCV RNA (to less than 500 copies per milliliter) within two months.

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