Biocompatible Anionic Polymeric Microspheres as Priming Delivery System for Effetive HIV/AIDS Tat-Based Vaccines

<div><p>Here we describe a prime-boost regimen of vaccination in <i>Macaca fascicularis</i> that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6P_cy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4⁺ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4⁺ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.</p></div

Quantitation of viral load upon intravenous virus challenge with SHIV89.6P_cy243 in control and vaccinated monkeys.

<p>In the left panels are reported the (<b>A</b>) plasma viremia and (<b>B</b>) the proviral DNA of control macaques. In the right panels are reported the (<b>C</b>) plasma viremia and the (<b>D</b>) proviral DNA of vaccinated macaques. In the bottom panels are indicated the trend line as a LOESS smoothed average of (<b>E</b>) plasma viral RNA and (<b>F</b>) proviral DNA of vaccinated (dashed line) and control (continous line) macaques. Statistical analyses were performed according to the Regression model for correlated data (means with 95% confidence limits).</p

Absolute numbers of CD4⁺ T cells following challenge with SHIV89.6P_cy243.

<p>The absolute CD4⁺ T cell counts are reported for (<b>A</b>) control and (<b>B</b>) vaccinated monkeys. In the left middle panel (<b>C</b>) the trend line as a LOESS smoothed average of the values of control (dashed line) and vaccinated (continous line) monkeys is shown. (<b>D</b>) Statistical analysis of the changes from baseline of CD4⁺ T cell counts in vaccinated and control macaques during the acute, post-acute and chronic phases of infection. The numbers within the panel indicate the level of statistically significant differences. (<b>E</b>) Analysis of the correlation of plasma viremia and CD4⁺ T cells counts in vaccinated (continuous red line) and control (black line) monkeys is reported.</p

Lymphoproliferative responses and frequency of cells producing IFN-γ, IL-2 or IL-4 during vaccination.

<p>The proliferative responses of (<b>A</b>) control and vaccinated (<b>B)</b> monkeys are reported as Stimulation Index (S.I). The dotted line indicated the cut-off of the assay. All samples showing a S.I. >3,0 were scored as reactive. In the lower panels the number of Spot Forming Cells (SFC/10⁶ PBMCs) of vaccinated macaques upon in vitro stimulation with Tat peptide pool are reported for the production of (<b>C</b>) IFN-γ, (<b>D</b>) IL-2 and (<b>E</b>) IL-4. None of the control macaques exhibited T cell responses and therefore the data are not presented. Arrows on the top of each panel indicate the time at which the vaccine antigen was given. The dashed line represents the cut-off (SFC/10⁶ PBMCs) of the assay. However, as described in the Material and Methods, were considered positive only samples yielding for IFN-γ ≥80 SFC/10⁶ cells and a fold increase ≥2.5; for IL-2≥30 SFC/10⁶ cells and a fold increase ≥3; for IL-4≥20 SFC/10⁶ cells and a fold increase ≥2.5.</p

Nonparametric Spearman rank analysis of the anti-Tat IgG subclass profile and anti-Tat T-cell responses (before challenge) in the vaccinated macaques.

<p>Nonparametric Spearman rank analysis of the anti-Tat IgG subclass profile and anti-Tat T-cell responses (before challenge) in the vaccinated macaques.</p

Summary of the virological and immunological status of “viremic” and “controller” vaccinated monkeys during the chronic phase of infection (weeks 22–74).

a<p>Monkey AG269 died at week 26 after challenge and was excluded from the analysis. Monkey AF134 died at week 46 after challenge.</p>b<p>According to their virological status during chronic infection, vaccinated monkeys were grouped as viremic (V) and Controllers (C). The numbers in parenthesis indicate the range (minimum-maximum value) of plasma viral RNA (Eq/ml). The p value indicates the statistical difference between the two groups.</p>c<p>Numbers in parenthesis indicate the range (minimum-maximum value) of CD4<b>⁺</b> T cell counts/mmc. The p value indicates the statistical difference between the two groups.</p>d<p>Numbers in parenthesis indicate the range (minimum-maximum value) of anti-Tat IgG antibody titers. The p value indicates the statistical difference between the two groups.</p>e<p>This monkey was negative for plasma viremia at 2 out 9 time points during the chronic infection.</p><p>Summary of the virological and immunological status of “viremic” and “controller” vaccinated monkeys during the chronic phase of infection (weeks 22–74).</p

Epitope mapping and virological outcome.

<p>(A) Plasma anti-Tat IgG responses to single Tat peptide were determined by ELISA at week 38 after the first immunization. To map specific responses, individual 15mer peptides (overlapping by 10 aa) spanning the aa 1–90 of Tat were used. The histogram inserted within the graph represents the anti-Tat IgG titers of vaccinated monkeys at week 38. (<b>B</b>) In the area plot graph is reported the cumulative means of the O.D. values of vaccinees grouped according to their virological status (controllers <b>in red</b> and viremics <b>in blue</b>). The dashed line with one, two or three asterisks represents the statistical analyses (Mann-Whitney test or unpaired t-test; GrapPad InStat vers 3.05 software, San Diego, Ca, USA) performed by comparing the epitope reactivity to the indicated Tat domains of controllers versus viremic macaques. These analyses were performed either comparing the reactivities to each single peptide (aa 61–75; * p = 0,037) or to grouped peptides (aa 61–90, *** p = 0,0084 and aa 61–85, ** p = 0,014). On the top of the graph are reported the Tat aa sequence and the associated Tat functional domains. The colored aa sequence represents the region against which anti-Tat antibodies from controllers but not viremic animals are directed.</p

Antibody responses against HIV-1 Env and Tat proteins after challenge with SHIV89.6P_cy243.

<p>Anti-Env IgG antibody titers were determined in plasma of control (<b>A</b>) and vaccinated (<b>B</b>) macaques. In the middle panels are reported the anti-Tat IgG antibody titers in (<b>C</b>) controls and (<b>D</b>) vaccinees. In the bottom panels the anti-Tat IgM antibody titers in (<b>E</b>) controls and (<b>F</b>) vaccinees are indicated. The dashed lines indicate the cut-off values (samples showing titers <1∶25 for IgM and <1∶100 for IgG were scored as negative).</p

Anti-Tat IgG subclass profile before challenge and virological outcome.

<p>(<b>A</b>) The box-blot analysis represents the anti-Tat IgG and IgG subclass profile in plasma of cynomolgus macaques before (week 23) and after (week 44) the last Tat/Alum boost. For the anti-Tat IgG subclass profile, a cut off for each subclass was established based on the analyses of IgG subclasses in plasma of 30 naive cynomolgus macaques, as described in material and methods. (<b>B</b>) The rate of plasma viral load decline among vaccinees is indicated where the lines (dashed red lines for viremic; continous black lines for controllers) represent the mobile median starting from the first positive viremia sample during the acute phase of the infection. (<b>C</b>) The impact of anti-Tat IgG1 titers on the rate of vRNA decline during the post acute phase of the infection, as determined at week 44 during the immunization, is reported.</p

Schedule of immunization and anti-Tat antibody responses in cynomolgus monkeys vaccinated with Tat/H1D and Tat/Alum.

<p>(<b>A</b>) Nine monkeys were injected intramuscularly with Tat/H1D microspheres at weeks 0, 4, 12 and 18, and boosted subcutaneously with the Tat in Alum at weeks 21 and 36, respectively. Nine control monkeys were primed with H1D alone boosted with Alum alone. (<b>B</b>) IgM and (<b>C</b>) IgG antibody titers in vaccinated monkeys. The arrows on the top of the each panel indicate the time at which the Tat/H1D, the Tat/Alum or H1D and Alum alone were given. The dashed lines indicate the cut-off values (samples showing titers <1∶25 for IgM and <1∶100 for IgG were scored as negative).</p