Gene map of recombinant NDV LaSota (rLaSota) and structures of gp160, gp120, gp140L, and gp140S.

<p>The proteins are shown as unprocessed primary translation products annotated to show the locations of the furin cleavage site, the membrane-proximal external region (MPER), the transmembrane (TM) and cytoplasmic (CT) domains, and the total aa lengths. The cDNAs encoding these proteins were modified by PCR to add NDV gene-start (GS) and gene-end (GE) transcription signals, an intergenic (IG) nucleotide, and flanking PmeI sites, and were inserted individually between the NDV P and M genes. Sequence flanking the Env ORFs is shown in positive-sense, and PmeI sites used in the construction are shown in italics. NDV genes (N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuraminidase protein; L, large polymerase protein) are shown as open boxes.</p

Oligomeric status of NDV-expressed Env proteins.

<p>DF1 cells were infected with indicated viruses at an MOI of 0.01 PFU. After 48 h, the cells were collected and cross-linked with DSP and subjected to SDS-PAGE under reducing (+R) or non-reducing (-R) conditions. The gels were analyzed by Western blotting using a pool of gp120-specific monoclonal antibodies. The positions of HIV-1 gp160, gp140 precursor gp120 are indicated by arrows in the right margin. Molecular masses of marker proteins (in kilodaltons) are shown in the left margin.</p

NDV-specific total IgG (panel A), and HIV-1 gp120-specific total IgG (panel B), IgG1 (panel C) and IgG2 (panel D) responses in guinea pig sera.

<p>The guinea pigs were immunized with the indicated rNDVs by the i.n. route. (A) The guinea pig sera were analyzed for NDV-specific antibodies by commercial NDV ELISA kits (Synbiotics Corporation). Mean ELISA end-point titers of NDV-specific serum antibodies on days 28 and 56 are shown. (B-D) The guine pig sera were analyzed by HIV-1 gp120 specific total IgG, IgG1 and IgG2 antibodies by isotype-specific ELISA with purified gp120. Mean ELISA end-point titers of gp120-binding serum antibodies of the indicated isotype on days 0, 7, 14, 21, 28, 42, 56, 70 and 90 are shown. Antibodies specific to gp120 were not detected in any animal on any day in the control rLaSota group. The graph shows the geometric mean value ± SEM for 3 animals in rLaSota, rLaSota/gp160 and rLaSota/gp140L groups, 6 animals in rLaSota/gp140S group and 5 animals in rLaSota/gp120 group. Arrows indicate time of rNDV immunizations on days 0 and 14. Statistical differences between the groups were calculated by unpaired t test (two-tailed). 1* indicates statistically significant differences (<i>P</i><0.05) of rLaSota/gp140S vs. rLaSota/gp160, rLaSota/gp140L and rLaSota/gp120 groups. 2* indicates statistically significant differences (<i>P</i><0.05) of rLaSota/gp140S vs. rLaSota/gp160 and rLaSota/gp140L groups.</p

Comparison of multicycle growth kinetics of rLaSota/gp160 (panel A), rLaSota/gp140L (panel B), rLaSota/gp140S (panel C), rLaSota/gp120 (panel D) viruses, each compared with the empty vector rLaSota virus.

<p>DF1 cells were infected with each virus at an MOI of 0.01 and cell culture media supernatant aliquots were harvested at 8 h intervals until 64 h post-infection. The virus titers in the aliquots were determined by plaque assay in DF1 cells. Values are averages from three independent experiments.</p

Detection of NDV-expressed Env proteins present in infected-cell lysates (A) and the cell culture medium (B).

<p>DF1 cells were infected with indicated viruses at an MOI of 0.01 PFU. After 48 h, the cell culture medium supernatants and cells were collected and processed. (A) The cell lysates were prepared from cells and subjected to SDS-PAGE under reducing conditions. (B) The cell culture medium supernatants were concentrated 10x by passing through Amicon filters and subjected to SDS-PAGE under reducing conditions. The gels were analyzed by Western blotting using a pool of gp120-specific monoclonal antibodies. The positions of HIV-1 gp160, gp140 precursor gp120 are indicated by arrows in the right margin. Molecular masses of marker proteins (in kilodaltons) are shown in the left margin.</p

HIV-1 gp120-specific total IgG (panel A), IgG1 (panel B), IgG2a (panel C) and IgA (panel D) antibodies in vaginal washes collected from guinea pigs, detected by isotype-specific ELISA with purified gp120.

<p>The guinea pigs were immunized with the indicated rNDVs by the i.n. route. Mean ELISA end-point titers of gp120-binding vaginal wash antibodies of the indicated isotype on days 0, 7, 14, 21, 28, 42, 56, 70 and 90 are shown. Antibodies specific to gp120 were not detected in any animal on any day in control rLaSota group. The graph shows the geometric mean value ± SEM for 3 animals in rLaSota, rLaSota/gp160 and rLaSota/gp140L groups, 6 animals in rLaSota/gp140S group and 5 animals in rLaSota/gp120 group. Arrows indicate time of rNDV immunizations on days 0 and 14. Statistical differences between the groups were calculated by unpaired t test (two-tailed). 1* indicates statistically significant differences (<i>P</i><0.05) of rLaSota/gp140S vs. rLaSota/gp160, rLaSota/gp140L and rLaSota/gp120 groups. 2* indicates statistically significant differences (<i>P</i><0.05) of rLaSota/gp140S vs. rLaSota/gp160 and rLaSota/gp140L groups.</p

Virus neutralizing antibody activity (50%-inhibitory-concentration [IC₅₀] titers) against homologous HIV-1 clade B tier 1 strains (BaL.26 and MN.3) and heterologous clade B tier 2 strains (RHPA4257.7, TRO.11, and SC22.3C2.LucR. T2A.ecto) in sera from guinea pigs immunized with the indicated rNDVs.

<p>(A) Guinea pig sera obtained on days 28, 56, 70 and 90 were tested against BaL.26 pseudovirus by the TZM-bl assay (B) Guinea pig sera obtained on days 28, 56, 70 and 90 were tested against MN.3 pseudovirus by the TZM-bl assay (C) Guinea pig sera obtained on day 56 were tested against RHPA4257.7 and TRO.11 by the TZM-bl assay, and against SC22.3C2.LucR. T2A.ecto by the A3R5 assay. Horizontal bars indicate the geometric mean titer. Pre-immune sera were used to establish baseline-neutralizing activity in each individual guinea pig, and these values were subtracted from the values shown. The neutralizing antibody activity against each virus in sera obtained from guinea pigs immunized with rLaSota virus was <20. Statistical differences between the groups were calculated by unpaired t test (two-tailed) and shown by the numbers underneath the horizontal line. * indicates statistically significant differences (<i>P</i><0.05) between groups.</p

Immunization schedules.

<p>A. Guinea pig immunization. Twenty seven guinea pigs were divided into 5 groups (n=6 for rLaSota/gp160, rLaSota/gp140L, rLaSota/gp140S, rLaSota/gp120 groups; n=3 for rLaSota group). Animals in each group were immunized with two doses of each recombinant virus on days 0 and 14 by i.n. route of administration. Each dose consisted of 300 µl (150 µl in each nostril) of allantoic fluid containing 10⁶ PFU/ml of virus. Blood, vaginal washes and fecal samples were collected on days 0, 7, 14, 21, 28, 42, 56, 70 and 90. All animals were sacrificed on day 90. B. Mice immunization. Thirty mice were divided in to 5 groups (n=6/group). Animals in each group were immunized with two doses of virus on days 0 and 14 by the i.n. route of administration. Each dose consisted of 50 µl (25 µl in each nostril) of allantoic fluid containing 10⁵ PFU/ml of virus. All animals were sacrificed on day 56 and splenocytes were collected.</p

HIV-1 Env-specific CD4+ (panel A) and CD8+ (panel B) T cell response.

<p>Mice in groups of 6 were immunized with 10⁵ PFU/ml of the indicated rNDV by the i.n. route on days 0 and 14. On day 56, splenocytes were isolated, stimulated with a pool of overlapping Env peptides, and processed for intracellular cytokine staining for IFN-γ and CD4 and CD8.</p

Multivalent Antigen Presentation Enhances the Immunogenicity of a Synthetic Three-Component HIV‑1 V3 Glycopeptide Vaccine

HIV-1 envelope glycoproteins gp120 and gp41 are presented on the virus surface as a trimer of heterodimer and are the targets of broadly neutralizing antibodies (bNAbs). We describe here the synthesis and preliminary immunological evaluation of a three-component trivalent HIV-1 V3 glycopeptide immunogen aiming to raise glycopeptide epitope-specific antibodies. Click chemistry confers efficient synthesis of the lipopeptide–glycopeptide conjugate that carries three copies of HIV-1 JR-FL gp120 V3 glycopeptide with a high-mannose glycan at the N332 glycosylation site. We found that the multivalent presentation substantially enhanced the immunogenicity of the V3 glycopeptide. The antisera induced by the three-component multivalent glycopeptide immunogen exhibited stronger binding to heterologous HIV-1 gp120s and the trimeric gp140s than that from the monovalent glycopeptide immunogen. The antisera generated from this preliminary rabbit immunization did not show virus neutralization activity, probably due to the lack of somatic maturation. The ability to elicit substantial glycopeptide epitope-specific antibodies by the three-component trivalent glycopeptide immunogen suggests that it could serve as a valuable vaccine component in combination with other vaccine candidates for further immunization studies