Absence of neutralising antibodies in early FIV infection – Group 2.

<p>Sequential plasmas from group 2 (825, 826, 827 and 828) animals collected at 0,3,6,10,12,15,18 and 21 weeks post-infection were diluted 1∶100 and assayed for the ability to neutralise HIV(FIV) pseudotypes bearing the B14, B19, B28, B30, B31 and B32 Envs. Also shown are pooled uninfected (−ve) plasma and post-mortem plasma from A613 (+ve). Neutralisation data are shown in comparison with total proviral loads for each cat as estimated using either <i>gag</i> or <i>env</i>–specific real-time PCR, or the variant (B14, B19, B28 and B30)-specific V5-targeted real-time PCR. All estimates of proviral load and luciferase activity are displayed as mean (n = 3) +/− SE.</p

Absence of neutralising antibodies in early FIV infection – Group 1.

<p>Sequential plasmas from group 1 (822, 823 and 824) animals collected at 0,3,6,10,12,15,18 and 21 weeks post-infection were diluted 1∶100 and assayed for the ability to neutralise HIV(FIV) pseudotypes bearing the B32 Env. Also shown are pooled uninfected (−ve) plasma and post-mortem plasma from A613 (+ve). Neutralisation data are shown in comparison with total proviral loads for each cat as estimated using either <i>gag</i> or <i>env</i>–specific real-time PCR. All estimates of proviral load and luciferase activity are displayed as mean (n = 3) +/− SE.</p

Cellular immune response in FIV infected cats.

<p>Cells from popliteal (PLN) or mesenteric (MLN) lymph nodes, peripheral blood (PBMC) or spleen were screened for interferon-<b>γ</b> production by ELISpot using AT2-inactivated virus as the source of viral antigen (each point represents the mean +/−SE of triplicate wells).</p

Epitope mapping of the cellular immune response in FIV infected.

<p>T cell epitopes were mapped using spleen cells from 822,823,824,825,826,827 and 828. Analyses used 17 pools of 10 x 15-mer peptides, each overlapping by 10 amino acids (pools P1-P17). Representative ELISpots for spleen cells from animal 822 against peptide pools P6 to p10) are included. All animals were screened with 17 peptide pools and where strong responses were confirmed (*), individual epitopes were then identified by splitting pools into their component peptides and repeating the analyses.</p

Serological response to FIV infection.

<p>Plasmas were collected from cats in group 1 (822, 823, 824) and group 2 (825, 826, 827 and 828) at 0, 3, 6, 10, 12, 15, 18 and 21 weeks post-infection and were screened by immunoblotting against purified GL8 virus. Also shown are positive (+) and negative (−) control plasma samples. Bands corresponding to matrix (MA) and capsid (CA) proteins, and the envelope glycoprotein (Env) are indicated by arrows.</p

Sensitivity of viral variants to receptor antagonists.

<p>Inhibitory concentration 50% (IC50) in µg/ml.</p

Proviral loads following FIV infection.

<p>Mean proviral loads (+/−SE) for groups 1 (clonal challenge) and 2 (quasispecies challenge) were estimated by real-time PCR using primers derived from either (A) <i>gag</i> (all clones shared a common <i>gagpol</i> sequence) or (B) a conserved sequence within the V5 region of <i>env</i>. (C) The mean proviral loads (+/−SE) for individual variants B14, B19, B28 and B30 in study group 2 were estimated using real-time PCR targeting unique determinants within the V5 region of <i>env</i>. All estimates of proviral load were performed in triplicate. (D) V5 <i>env</i> sequences amplified at 21 weeks post-infection from cats 825, 826, 827 and 828 infected with the reconstituted quasispecies. The region spanning nucleotides 1640 to 1694 of the GL8 <i>env</i> open reading frame is shown, the predicted amino acid translation of this region from the parent GL8 strain is shown above the parent GL8 sequence. The number of clones isolated with the corresponding V5 sequence is listed; no clones bore the B14 sequence, consistent with the poor replication of B14 <i>in vivo</i>.</p

(A) Location of T cell epitopes identified by ELISpot on schematic representations of FIV gp120 and gp41.

<p>Each circle represents a single amino acid, regions constituting T cell epitopes are marked by a red line with responding cat number alongside and peptide names in parenthesis. Predicted sites for N-linked glycosylation are in blue. (B) Amino acid sequences of T cell epitopes covered by the ELISpot peptides and the animals that responded.</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

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