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. 2012;7(2):e31608.
doi: 10.1371/journal.pone.0031608. Epub 2012 Feb 15.

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates

Paula Ordonhez Rigato  1 , Milton Maciel Jr, Adriana Letícia Goldoni, Orlando Guerra Piubelli, Noemia Mie Orii, Ernesto Torres Marques, Joseph Thomas August, Alberto José da Silva Duarte, Maria Notomi Sato
Affiliations

Maternal LAMP/p55gagHIV-1 DNA immunization induces in utero priming and a long-lasting immune response in vaccinated neonates

Paula Ordonhez Rigato et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Transfer of high levels of anti-Gag IgG1 Abs from Lamp/gag DNA-immunized mothers to offspring through transplacental and breastfeeding routes.
Female BALB/c mice were primed and boosted with 50 µg of Lamp (L), gag (G) or Lamp/gag (LG) plasmid DNA and mated one day after the boost. (A) Maternal serum and (B) amniotic fluid were obtained by caesarean section from full-term pregnant mice. (C) Breast-milk samples (1∶20) were collected on day 5 after delivery and (D) fetal serum from 0-, 30-, 60- and 90-d-o offspring were evaluated by ELISA using HIV-1 lysates. The results from 4–6 animals per group were expressed as the mean ± SEM. Schematic diagram of the immunization protocol is shown. *p≤0.05 and ***p<0.001 when compared to gag.
Figure 2
Figure 2. Maternal immunization with the Lamp/gag DNA vaccine decreases anti-Gag IgG1 Ab production in immunized offspring.
Female mice were immunized, and 20 days later, they were boosted with 50 µg Lamp (L), gag (G), or Lamp/gag (LG) plasmid DNA and mated one day after the boost. Offspring were either non-immunized or immunized with 5 µg of the same vaccine DNA at 7- and 25-d-o. Offspring serum samples were collected at (A) 30- and (B) 60-d-o (3–8 animals per group). Anti-Gag IgG1 was determined by ELISA using HIV-1 lysates. Bars represent the mean ± SEM. Schematic diagram of the immunization protocols are shown. *p≤0.05 and ***p<0.001 when compared to the respective control offspring from non-immunized (NI) mothers.
Figure 3
Figure 3. Maternal Lamp/gag immunization reduces the IFN-γ response of immunized offspring.
Offspring from mothers immunized with Lamp/gag (LG) or gag (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.
Figure 4
Figure 4. Effect of maternal DNA immunization on TGF-β1 levels in milk and on the generation of CD4+CD25+ FoxP3+ T cells in offspring.
Female mice were primed and boosted with 50 µg of Lamp/gag (LG), gag (G), or Lamp (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 gag-immunized group.
Figure 5
Figure 5. Transfer of IgG from immune mice decreases CTL numbers and anti-Gag IgG Abs in offspring immunized with LAMP/gag.
Offspring from non-immunized mothers were immunized at 7- and 25-d-o with 5 µg of LAMP/gag (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) In vivo 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.
Figure 6
Figure 6. Maternal LAMP/gag immunization does not interfere with long-lasting IFN-γ and B-cell responses in immunized offspring.
Offspring from non-immune (NI) or immune (I) mothers were immunized at 7- and 25-d-o with 5 µg of either Lamp/gag (LG) or gag (G) DNA. Some neonates groups from I or NI mothers received a 3rd boost at 6-months-old. (A) IFN-γ SFCs from 6-month-old offspring after stimulation with MHC H-2d class I- and II-restricted pools of Gag peptides from HIV-1. The figure represents 4–5 assays per group (2–3 animals per group); *p≤0.05 when compared with immune offspring from NI mothers. (B) Serum from offspring was collected when they were 6 months old (6 animals per group), and anti-Gag IgG was evaluated by ELISA using HIV-1 lysates. One group received a boost 10 days before analysis. Bars represent the mean ±SEM. *p≤0.05 when compared with immune offspring from NI mothers. Schematic diagram of the immunization protocols are shown **p<0.01 when compared with gag-immunized offspring.
Figure 7
Figure 7. Intravenous DNA immunization during pregnancy primes the fetal immune system.
Pregnant mice were IV immunized approximately 15–16 days into gestation with 100 µg of Lamp/gag (LG) or gag (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.
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