A single lentivector DNA based immunization contains a late heterologous SIVmac251 mucosal challenge infection

Variety of conventional vaccine strategies tested against HIV-1 have failed to induce protection against HIV acquisition or durable control of viremia. Therefore, innovative strategies that can induce long lasting protective immunity against HIV chronic infection are needed. Recently, we developed an integration-defective HIV lentiDNA vaccine that undergoes a single cycle of replication in target cells in which most viral antigens are produced. A single immunization with such lentiDNA induced long-lasting T-cell and modest antibody responses in cynomolgus macaques. Here eighteen months after this single immunization, all animals were subjected to repeated low dose intra-rectal challenges with a heterologous pathogenic SIVmac251 isolate. Although the viral set point in SIVmac-infected cynomolgus is commonly lower than that seen in Indian rhesus macaques, the vaccinated group of macaques displayed a two log reduction of peak of viremia followed by a progressive and sustained control of virus replication relative to control animals. This antiviral control correlated with antigen-specific CD4+ and CD8+ T cells with high capacity of recall responses comprising effector and central memory T cells but also memory T cell precursors. This is the first description of SIV control in NHP model infected at 18 months following a single immunization with a non-integrative single cycle lentiDNA HIV vaccine. While not delivering sterilizing immunity, our single immunization strategy with a single-cycle lentivector DNA vaccine appears to provide an interesting and safe vaccine platform that warrants further exploration

In Vivo Administration of a JAK3 Inhibitor during Acute SIV Infection Leads to Significant Increases in Viral Load during Chronic Infection

<div><p>The studies reported herein are the first to document the effect of the in vivo administration of a JAK3 inhibitor for defining the potential role of NK cells during acute SIV infection of a group of 15 rhesus macaques (RM). An additional group of 16 MHC/KIR typed RM was included as controls. The previously optimized in vivo dose regimen (20 mg/kg daily for 35 days) led to a marked depletion of each of the major NK cell subsets both in the blood and gastro-intestinal tissues (GIT) during acute infection. While such depletion had no detectable effects on plasma viral loads during acute infection, there was a significant sustained increase in plasma viral loads during chronic infection. While the potential mechanisms that lead to such increased plasma viral loads during chronic infection remain unclear, several correlates were documented. Thus, during acute infection, the administration of the JAK3 inhibitor besides depleting all NK cell subsets also decreased some CD8⁺ T cells and inhibited the mobilization of the plasmacytoid dendritic cells in the blood and their localization to the GIT. Of interest is the finding that the administration of the JAK3 inhibitor during acute infection also resulted in the sustained maintenance during chronic infection of a high number of naïve and central memory CD4⁺ T cells, increases in B cells in the blood, but decreases in the frequencies and function of NKG2a⁺ NK cells within the GIT and blood, respectively. These data identify a unique role for JAK3 inhibitor sensitive cells, that includes NK cells during acute infection that in concert lead to high viral loads in SIV infected RM during chronic infection without affecting detectable changes in antiviral humoral/cellular responses. Identifying the precise mechanisms by which JAK3 sensitive cells exert their influence is critical with important implications for vaccine design against lentiviruses.</p></div

Aliquots of plasma samples from each of the 16 control monkeys and the 15 monkeys that received the JAK3 inhibitor were assayed for viral loads post SIVmac239 infection.

<p>The # of viral copies/ml of plasma from A) the control monkeys and B) the monkeys that received the JAK3 inhibitor are shown. The data in C) reflects the same data expressed as dot plots with means and D) illustrates these data as geometric means. (ns = non significant).</p

The absolute number of total (A & B), naïve (C & D) central memory (E & F) and effector memory (G & H) CD4⁺ T cells in the PBMCs of the 2 group of animals expressed as dot plots (A, C, E and G) and geometric means (B, D, F & H) is shown.

<p>While there was no significant difference in the numbers of effector memory CD4⁺ T cells between the 2 groups of animals, there was clearly a significant difference in the total (p<0.016), naïve (p<0.008) and central memory CD4⁺ T cells (p<0.016).</p

SIV specific antibody titers (A & B) calculated as O.D₄₅₀ and ADCC titers calculated as IC₅₀ values (C &D) in the plasma of the 2 groups of monkeys is illustrated as dot plots (A, C) or geometric means (B & D).

<p>In addition, the frequency of the p11C (C–M) tetramer⁺ cells as a function of the gated population of CD3⁺, CD8⁺ T cells are shown as a dot plot (E) or geometric means (F). The differences were not significant (n.s.).</p

Aliquots of the mononuclear cells isolated from pools of GIT biopsies from the same animals as described under Figure 1 post SIVmac239 infection were used to isolate DNA and subjected to PCR using conserved SIVgag specific primer pairs.

<p>Data illustrated shows # copies/ng DNA from A) Control animals B) Animals that received the JAK3 inhibitor C) reflects the same data shown as dot plots with means and D) illustrates these data as Median values. The median values were chosen instead of Geometric means in this case because the formulae used to derive Geometric means cannot include negative or zero values. The symbols (***) represent p<0.0005 and (**) represents p<0.004 values.</p

The efficiency (% change from a mean of 3 baseline values) of depletion in the absolute numbers of CD3⁻, CD8α⁺, NKG2a⁺ (total NK cells) in the PBMC of control and the animals that received the JAK3 inhibitor as a function of time post infection is illustrated.

<p>A) Shows the data with Means +/− SD and B) the Geometric means for the 2 groups of animals. The difference in the values at 2 weeks post infection in the 2 groups of animals was highly significant (p<0.0001).</p

Functional assessment of NK cells in an aliquot of the PBMC from each of the 2 groups of animals was determined by measuring the frequencies of NK cells expressing CD107 upon co-culture with the 721.221 cell line as described in the methods section.

<p>The frequencies of CD107⁺ cells are expressed as dot plots with means (A) or geometric means (B). Aliquot of the same cells were also assessed for undergoing apoptosis in the presence and absence of soluble CD95 as described in the methods section. Data of the frequency of Annexin V expressing NKG2a⁺ cells is shown as dot plots (C) or geometric means (D). The differences in the re-emergence of NK cell function reflected as the frequencies of CD107+ NK cells in 4/16 control animals that spontaneously controlled plasma viremia (n = 2) and those with low viral loads (n = 2, LVL) as compared with those with high viral load (n = 12, HVL) is shown in the inset of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003929#ppat-1003929-g008" target="_blank">Fig. 8 B</a>.</p

The absolute number of total CD8⁺ T cells (A & B), total B cells denoted as CD20 (C & D) and plasmacytoid dendritic cells denoted as pDC (E & F) in the PBMCs from the 2 groups of animals is illustrated as dot plots with means (A, C & E) and geometric means (B, D & F).

<p>The statistical differences are noted as either n.s. (Not significant) or with appropriate p values.</p

The absolute numbers of NK cells and its subsets within the PBMC of the 2 group of animals is illustrated as dot plots with means (A, C, E, G & I) and Geometric means (B, D, F, H & J) of total CD3⁻, CD8α⁻, NKG2a⁺ NK cells (A, B) and the CD16⁺, CD56⁻ cytolytic subset (C, D), the cytokine sythesizing subset CD16⁻, CD56⁺ (E, F), the CD16⁺, CD56⁺ (G, H) and the CD16⁻, CD56⁻ (I, J) subset.

<p>Please note the NK cell lineage that rebounds first (Inset <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003929#ppat-1003929-g004" target="_blank">Figure 4F</a>).</p