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

Immunogenicity of LAMP-1/p55Gag of HIV-1 DNA chimeric vaccine on neonatal period and effect of maternal immunization immune response of vaccinated offspring.

A imunogenicidade da vacina de DNA LAMP/gag do HIV-1, que direciona o antígeno aos compartimentos contendo moléculas de classe II do MHC, foi analisada em camundongos BALB/c neonatos e também foi analisado o efeito da imunização materna na resposta vacinal da prole. A vacina LAMP/gag neonatal foi capaz de induzir a produção de anticorpos IgG anti-Gag, aumentar o número de células produtoras de IFN-g para epítopos de classe I e II do MHC, células T CD8+ (TNF-a, IFN-g, granzima) e específicas ao epítopo imunodominante de classe I do MHC da Gag. Além de gerar resposta humoral e celular de longa duração observado até os 9 meses de idade. A imunização pré-concepcional com LAMP/gag transfere anticorpos IgG1 anti-Gag às proles (via transplacentária e leite), diminui a resposta humoral/celular, induz células CD4+CD25+FoxP3, mas não interfere na geração da resposta de memória da prole imunizada. Estrategicamente, a imunização por via intravenosa com LAMP/gag durante a gestação evita a transferência de anticorpos e é capaz de sensibilizar o sistema imunológico fetal.The immunogenicity of LAMP/gag DNA vaccine of HIV-1, which target the antigen to the class II MHC compartment, was evaluated on BALB/c neonate mice and also the effect of maternal immunization on off spring vaccine response was analyzed. The LAMP/gag neonatal vaccination elicited IgG antibody anti-Gag production and augment the IFN-g producing cells to the MHC class I and II epitopes; CD8+ T cells expressing TNF-a, IFN-g, granzime and class I immunodominant epitope of Gag specific cells. Moreover of generate long lasting humoral and cellular immune response until 9 months of age. The LAMP/gag pre-conceptional immunization transferred IgG1 antibody anti-Gag to offspring (by transplacentary and breastmilk route), diminished humoral/cellular response, inducing CD4+CD25+FoxP3 cells on immunized offspring, however did not affect the memory response. Strategically, the intravenous immunization during gestation avoided the antibody transference and primed the fetal immune system

Mucosal and systemic anti-GAG immunity induced by neonatal immunization with HIV LAMP/gag DNA vaccine in mice

Vaccines capable of inducing mucosal immunity in early postnatal life until adulthood, protecting early sexual initiation, should be considered as strategies to vaccination against HIV. The HIV-1 GAG protein as a chimera with the lysosome-associated membrane protein (LAMP/gag), encoded by a DNA vaccine, is targeted to the endosomal/lysosomal compartment that contains class II MHC molecules and has been shown to be immunogenic in adult mice. Assuming that one such strategy could help to overcome the immunological immaturity in the early postnatal period, we have evaluated the systemic and mucosal immunogenicity of LAMP/gag immunization in neonatal mice. Intranasal immunization with LAMP/gag vaccine induced higher levels of sIgA and IgG anti-GAG antibodies in intestinal washes than did the gag vaccine. The combination of ID injections and the IN protocol with the chimeric vaccine promoted the increase of Ab levels in sera. Both vaccines induced splenic IFN-gamma- secreting cells against GAG peptide pools, as well as in vivo cytotoxic T lymphocyte (CTL) function, and increased the percentage of CD8+ T cells to the immunodominant class I peptide in gut and spleen. However, only the chimeric vaccine was able to enhance Th1/Th2 cytokine secretion in response to class II GAG peptide and to enhance IL-4-secreting cells against GAG peptides and p24 protein stimuli. Long-lasting humoral and cellular responses were detected until adult age, following neonatal immunization with the chimeric vaccine. The LAMP/gag vaccination was able to induce potent GAG-specific T and B cell immune responses in early life which are essential to elicit sustained and long-lasting mucosal and systemic humoral response. (C) 2010 Elsevier GmbH. All rights reserved.Ministerio da Saude do Brasil - Programa Nacional de HIV/AIDS/DST[914BRA1101]FAPESPUniversidade de São Paulo - LIM-56/HCFMUS

The Dermatophyte <i>Trichophyton rubrum</i> Induces Neutrophil Extracellular Traps Release by Human Neutrophils

Neutrophils are the first leukocytes recruited to the site of infection and are thought to be responsible for fungal elimination from the skin such as dermatophytes. Neutrophils are able to secrete reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) that can kill different fungi, including Aspergillus, spp., Candida albicans, and Phialophora verrucosa. However, NET production in response to Trichophyton rubrum, the main etiologic agent of dermatophytosis, has yet to be studied. We demonstrated that human neutrophils produce NETs against different morphotypes of T. rubrum in a dose-dependent manner and NET formation is dependent on ROS production. In addition, ROS production by human neutrophils in response to T. rubrum is dependent on NADPH oxidase, but not on fungal viability. NETs mediated killing of T. rubrum. Collectively, these results demonstrate that T. rubrum was able to trigger the production of NETs, suggesting that these extracellular structures may represent an important innate immune effector mechanism controlling physiological response to T. rubrum infection

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-gamma, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric IAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory. (C) 2010 Elsevier Inc. All rights reserved.CNPq[141487/2005]FAPESP[2004/14443-2]Ministerio da Saude do Brasil[914BRA1101]Universidade de São Paulo - LIM-56/HC-FM-US

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

LAMP-1 Chimeric to HIV-1 p55Gag in the Immunization of Neonate Mice Induces an Early Germinal Center Formation and AID Expression

Neonates have a limited adaptive response of plasma cells, germinal center (GC) B cells, and T follicular helper cells (TFH). As neonatal vaccination can be an important tool for AIDS prevention, these limitations need to be overcome. Chimeric DNA vaccine encoding p55Gag HIV-1 protein conjugated with lysosomal-associated membrane protein 1 (LAMP-1) has been described as immunogenic in the neonate period. Herein, we investigated the immunologic mechanisms involved in neonatal immunization with a LAMP-1/p55Gag (LAMP/Gag) DNA vaccine in a C57BL/6 mouse background. Neonatal LAMP/Gag vaccination induced strong Gag-specific T-cell response until adulthood and elevated levels of anti-Gag IgG antibodies. We also demonstrated for the first time that the immunogenicity of the neonatal period with LAMP/Gag is due to the induction of high-affinity anti-p24 IgG antibodies and long-term plasma cells. Together with that, there is the generation of early TFH cells and the formation of GC sites with the upregulation of activation-induced cytidine deaminase (AID) enzyme mRNA and protein expression in draining lymph nodes after neonatal LAMP/Gag vaccination. These findings underscore that the LAMP-1 strategy in the chimeric vaccine could be useful to enhance antibody production even in the face of neonatal immaturity, and they contribute to the development of new vaccine approaches for other emerging pathogens at an early stage of life

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

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