Maternal LAMP/p55gagHIV-1 DNA Immunization Induces In Utero Priming and a Long-Lasting Immune Response in Vaccinated Neonates

Infants born to HIV-infected mothers are at high risk of becoming infected during gestation or the breastfeeding period. A search is thus warranted for vaccine formulations that will prevent mother-to-child HIV transmission. The LAMP/gag DNA chimeric vaccine encodes the HIV-1 p55gag fused to the lysosome-associated membrane protein-1 (LAMP-1) and has been shown to enhance anti-Gag antibody (Ab) and cellular immune responses in adult and neonatal mice; such a vaccine represents a new concept in antigen presentation. In this study, we evaluated the effect of LAMP/gag DNA immunization on neonates either before conception or during pregnancy. LAMP/gag immunization of BALB/c mice before conception by the intradermal route led to the transfer of anti-Gag IgG1 Ab through the placenta and via breastfeeding. Furthermore, there were an increased percentage of CD4+CD25+Foxp3+T cells in the spleens of neonates. When offspring were immunized with LAMP/gag DNA, the anti-Gag Ab response and the Gag-specific IFN-γ-secreting cells were decreased. Inhibition of anti-Gag Ab production and cellular responses were not observed six months after immunization, indicating that maternal immunization did not interfere with the long-lasting memory response in offspring. Injection of purified IgG in conjunction with LAMP/gag DNA immunization decreased humoral and cytotoxic T-cell responses. LAMP/gag DNA immunization by intradermal injection prior to conception promoted the transfer of Ab, leading to a diminished response to Gag without interfering with the development of anti-Gag T- and B-cell memory. Finally, we assessed responses after one intravenous injection of LAMP/gag DNA during the last five days of pregnancy. The intravenous injection led to in utero immunization. In conclusion, DNA vaccine enconding LAMP-1 with Gag and other HIV-1 antigens should be considered in the development of a protective vaccine for the maternal/fetal and newborn periods

Transfer of IgG from immune mice decreases CTL numbers and anti-Gag IgG Abs in offspring immunized with <i>LAMP/gag</i>.

<p>Offspring from non-immunized mothers were immunized at 7- and 25-d-o with 5 µg of <i>LAMP/gag</i> (LG) and treated with IgG from non-immune (IgG-NI) or LG (IgG-LG)-immunized mice at 7-, 13-, and 25-d-o or without IgG treatment (none). (A) Offspring serum samples from 35-d-o mice (n = 6 per group) were evaluated for anti-Gag IgG Ab levels by ELISA using HIV-1 lysates. (B) <i>In vivo</i> T-cell cytotoxicity was evaluated ten days after LG immunization. Mice were IV injected with target cells from NI mice stained with low and high concentrations of CFSE. The CFSE-high target cells were pulsed with a class I immunodominant peptide (AMQMLKETI), and after 18 h, spleen cells were evaluated. (C) IFN-γ SFCs from 35-d-o offspring (n = 3 per group) were evaluated after stimulation with Gag MHC class I and II epitopes or recombinant p55 of HIV-1. Bars represent the mean ± SEM. Schematic diagram of the immunization protocol is shown. **p≤0.01 compared with offspring treated with IgG from NI mice; #p≤0.05 compared with offspring without IgG treatment.</p

Maternal <i>Lamp/gag</i> immunization reduces the IFN-γ response of immunized offspring.

<p>Offspring from mothers immunized with <i>Lamp/gag</i> (LG) or <i>gag</i> (G) were immunized with 5 µg of (A) LG or (B) G DNA, respectively. Spleen cells from 35-d-o offspring were cultured with 25 pooled HIV-1 Gag peptides. The figure represents 4–5 assays per group (2–3 animals per group). Bars show the mean ± SEM. Schematic diagram of the immunization protocols are shown. *p≤0.05 and **p<0.01 when compared with immunized offspring from non-immunized (NI) mothers.</p

Effect of maternal DNA immunization on TGF-β1 levels in milk and on the generation of CD4+CD25+ FoxP3+ T cells in offspring.

<p>Female mice were primed and boosted with 50 µg of <i>Lamp/gag</i> (LG), <i>gag</i> (G), or <i>Lamp</i> (L) plasmid DNA and mated one day after the boost. (A) Breast-milk samples collected 7 days after delivery; (B) percentages of CD4+CD25+Foxp3+ T cells in offspring splenocytes at 7 d-o; (C) CD4+CD25+FoxP3+ T cells in individual offspring at 7 d-o, non-immunized (NI), or immunized with L, G or LG. The results of 8–12 animals per group are expressed as the mean ± SEM. Schematic diagram of the immunization protocol is shown. #p≤0.05 and ##p<0.01 when compared with the non-immunized (NI) group; **p<0.01 when compared with the <i>gag</i>-immunized group.</p

Intravenous DNA immunization during pregnancy primes the fetal immune system.

<p>Pregnant mice were IV immunized approximately 15–16 days into gestation with 100 µg of <i>Lamp/gag</i> (LG) or <i>gag</i> (G) vaccine DNA. Offspring were either immunized (IM) or not (NI) at 7-d-o with 5 µg of the DNA vaccine. (A) Mother: IFN-γ SFCs from spleen cells of immunized mothers 35 days after delivery and (B) Offspring: IFN-γ SFCs from spleen cells of 35-d-o offspring after stimulation with class I and class II Gag peptides and recombinant p24 of HIV-1. The figure represents 2–3 assays per group (2–3 animals per group). (C) Offspring: Serum samples from 35-d-o NI or IM offspring were evaluated by ELISA using HIV-1 lysates for anti-Gag IgG concentrations. Bars represent the mean ± SEM. Schematic diagram of the immunization protocol is shown. *p≤0.05, **p<0.01, and *** p<0.001 when compared with G-offspring from a G-mother; +++p<0.001 when compared with NI offspring from a G-immunized mother.</p