Differences in Shannon entropy between controls and vaccinees.

<p>The Shannon entropy was calculated at each nucleotide position for pooled vaccinees, group 1 vaccinees and group 3 vaccinees and subtracted from the entropy calculated for control animals. Sites displaying a statistically significant difference in entropy between controls and vaccinees are indicated in red.</p

Positions showing statistically significant differences in Shannon entropy between controls and vaccinees.

<p>The sequences of all vaccinees were compared against the sequences of all control animals or the sequences of vaccines were divided according to the vaccination protocol (groups 1 and 2) and compared against controls. The last column shows the nucleotide sites-of-interest identified using a Bayesian model.</p><p>CT: cytoplasmic tail.</p><p>no: sites not identified by the Bayesian tool.</p

A) Maximum likelihood phylogenetic tree of the envelope gene of the virus inoculum.

<p>The scale bar represents one nucleotide substitution. <b>B) Frequency of each inoculum variant at the amino acid level.</b> The number of each viral genome present within the inoculum was estimated based on the amino acid sequence (black bars) and at the nucleotide level (grey bars).</p

T-cell ELISpot responses 2 weeks after the last immunization.

<p>Positive IFN-γ, IL-2 and IL-4 ELISPots per million cells are shown for each animal after stimulation with full-length recombinant proteins of clades A, B and C and V2-V3 peptides of clades B and C once 2 background responses were subtracted. Positive responses are defined as responses above 50 spot forming units per million cells.</p

Composite phylogenetic tree of all envelope sequences along with sequences of the challenge inoculum.

<p>Master phylogeny shows no clustering according to vaccination status. Insert shows representative intrahost phylogenies. Animal-derived sequences are color-coded. Black circles indicate inoculum-derived sequences. The scale bar indicates one nucleotide substitution.</p

Protein divergence between animal sequences and vaccine inserts.

<p>The mean protein divergence from the protein sequences included in the vaccine regime (gp140 in A and peptides used in boosting in B) and standard deviation are reported. Only statistically significant comparisons are indicated. C) The reciprocal of serum dilutions at which 50% inhibition of viral infection (IC₅₀) occurred from sera derived from the day of challenge from animals that were protected (left) or protected and non-protected (right) against pseudotypes harbouring Envs derived from the virus inoculum or animal plasma (founder Envs) were compared. No statistically significant differences were identified (un-paired t-test).</p

Pre-challenge neutralizing antibody responses.

<p>Sera collected at different time points before and after challenge were tested against single-round competent virus bearing an Env protein of the SHIV_sf162p4 virus inoculum. The reciprocal of serum dilutions at which 50% inhibition of viral infection (IC₅₀) occurred are reported.</p

Trial outcome.

<p><b>A) Schematic representation of the experimental design. B) Infection kinetics after mucosal challenge with SHIV_SF162P4.</b> Viral loads were measured at the indicated times post infection for a period of 12 weeks in each of the experimental groups. Since the detection level of the assay was 100 viral copies/ml negative samples were assigned a value of 2 (Log10 of 100). <b>C) Distribution of peak viral RNA loads.</b> Continuous lines indicate the means of each group; upper discontinuous line indicates viremia 1 log lower than the mean peak viremia of control animals while lower line indicates the limit of detection of the assay (100 viral RNA copies). Control animal 96015 was not included in the graphic and calculations since its viral load continue to increase during the study period.</p

Binding antibodies after the last immunization.

<p>The presence of binding antibodies against gp140 or the indicated peptides used for immunization was measured using ELISA at a single serum dilution. The sera collected after 2 weeks of the last immunization was analysed against gp140 while the sera collected 6 weeks the last immunization was analysed against the indicated peptides. The interrupted lines depict the threshold OD value above which a sample is considered positive (3 times the OD value of the pre-immunization sera).</p

Genetic Imprint of Vaccination on Simian/Human Immunodeficiency Virus Type 1 Transmitted Viral Genomes in Rhesus Macaques

Understanding the genetic, antigenic and structural changes that occur during HIV-1 infection in response to pre-existing immunity will facilitate current efforts to develop an HIV-1 vaccine. Much is known about HIV-1 variation at the population level but little with regard to specific changes occurring in the envelope glycoprotein within a host in response to immune pressure elicited by antibodies. The aim of this study was to track and map specific early genetic changes occurring in the viral envelope gene following vaccination using a highly controlled viral challenge setting in the SHIV macaque model. We generated 449 full-length env sequences from vaccinees, and 63 from the virus inoculum. Analysis revealed a different pattern in the distribution and frequency of mutations in the regions of the envelope gene targeted by the vaccine as well as different patterns of diversification between animals in the naïve control group and vaccinees. Given the high stringency of the model it is remarkable that we were able to identify genetic changes associated with the vaccination. This work provides insight into the characterization of breakthrough viral populations in less than fully efficacious vaccines and illustrates the value of HIV-1 Env SHIV challenge model in macaques to unravel the mechanisms driving HIV-1 envelope genetic diversity in the presence of vaccine induced-responses