Milton Maciel

Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

Oral priming with Salmonella Typhi vaccine strain CVD 909 followed by parenteral boost with the S. Typhi Vi capsular polysaccharide vaccine induces CD27+ IgD−S. Typhi specific IgA and IgG B memory cells in humans

Attenuated live oral typhoid vaccine candidate CVD 909 constitutively expresses Salmonella Typhi capsular polysaccharide antigen (Vi). A randomized, double-blind, heterologous prime-boost clinical study was conducted to determine whether immunity to licensed parenteral Vi vaccine could be enhanced by priming with CVD 909. Priming with CVD 909 elicited higher and persistent, albeit not significant, anti-Vi IgG and IgA following immunization with Vi, than placebo-primed recipients. Vi-specific IgA B memory (B(M)) cells were significantly increased in CVD 909-primed subjects. S. Typhi-specific LPS and flagella IgA B(M) cells were observed in subjects immunized with CVD 909 or with the licensed Vi-negative oral typhoid vaccine Ty21a. CVD 909-induced B(M) cells exhibited a classical B(M) phenotype (i.e., CD3(-)CD19(+)IgD(-)CD27(+)). This is the first demonstration of classical B(M) cells specific for bacterial polysaccharide or protein antigens following typhoid immunization. The persistent IgA B(M) responses demonstrate the capacity of oral typhoid vaccines to prime mucosally relevant immune memory.

DNA Encoding an HIV-1 Gag/Human Lysosome-Associated Membrane Protein-1 Chimera Elicits a Broad Cellular and Humoral Immune Response in Rhesus Macaques

Previous studies of HIV-1 p55Gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type II (MHC II) complex molecules by use of a DNA antigen formulation encoding Gag as a chimera with the mouse lysosome-associated membrane protein (mLAMP/gag). In the present study, we have analyzed the magnitude and breadth of Gag-specific T-lymphocyte and antibody responses elicited in Rhesus macaques after immunization with DNA encoding a human LAMP/gag (hLAMP/gag) chimera. ELISPOT analyses indicated that the average Gag-specific IFN-γ response elicited by the hLAMP/gag chimera was detectable after only two or three naked DNA immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (SFC)/106 PBMCs. High IFN-γ ELISPOT responses were detected in CD8+-depleted cells, indicating that CD4+ T-cells play a major role in these responses. The T-cell responses of four of the macaques were also tested by use of ELISPOT to 12 overlapping 15-amino acids (aa) peptide pools containing ten peptides each, encompassing the complete Gag protein sequence. The two Mamu 08 immunized macaques responded to eight and twelve of the pools, the Mamu B01 to six, and the other macaque to five pools indicating that the hLAMP/gag DNA antigen formulation elicits a broad T-cell response against Gag. Additionally, there was a strong HIV-1-specific IgG response. The IgG antibody titers increased after each DNA injection, indicating a strong amnestic B-cell response, and were highly elevated in all the macaques after three immunizations. Moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete Gag amino acid sequence. In addition, HIV-1-specific IgA antibodies were present in the plasma and external secretions, including nasal washes. These data support the findings of increased immunogenicity of genetic vaccines encoded as LAMP chimeras, including the response to DNA vaccines by non-human primates.

Antigen-specific B memory cell responses to lipopolysaccharide (LPS) and invasion plasmid antigen (Ipa) B elicited in volunteers vaccinated with live-attenuated Shigella flexneri 2a vaccine candidates.

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B(M) cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10(6) expanded cells following vaccination (p=0.008). A strong correlation was found between post-vaccination anti-LPS B(M) cell counts and peak serum anti-LPS IgG titers (rs=0.95, p=0.0003). Increases in B(M) specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B(M) cells to LPS and IpaB, suggesting that B(M) responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.

Antigen-specific IgA B memory cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates

We studied the induction of antigen-specific IgA memory B cells (B(M)) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 10(7), 10(8) or 10(9) CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA B(M) cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA B(M) cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA B(M) cells and anti-LPS IgA in serum and stool; IgA B(M) cell responses to IpaB were also observed. These B(M) cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response.

Comprehensive analysis of T cell epitope discovery strategies using 17DD yellow fever virus structural proteins and BALB/c (H2d) mice model.

Abstract Immunomics research uses in silico epitope prediction, as well as in vivo and in vitro approaches. We inoculated BALB/c (H2d) mice with 17DD yellow fever vaccine to investigate the correlations between approaches used for epitope discovery: ELISPOT assays, binding assays, and prediction software. Our results showed a good agreement between ELISPOT and binding assays, which seemed to correlate with the protein immunogenicity. PREDBALB/c prediction software partially agreed with the ELISPOT and binding assay results, but presented low specificity. The use of prediction software to exclude peptides containing no epitopes, followed by high throughput screening of the remaining peptides by ELISPOT, and the use of MHC-biding assays to characterize the MHC restrictions demonstrated to be an efficient strategy. The results allowed the characterization of 2 MHC class I and 17 class II epitopes in the envelope protein of the YF virus in BALB/c (H2d) mice.

Human Leukocyte Antigen (HLA) Class I Restricted Epitope Discovery in Yellow Fewer and Dengue Viruses: Importance of HLA Binding Strength

Epitopes from all available full-length sequences of yellow fever virus (YFV) and dengue fever virus (DENV) restricted by Human Leukocyte Antigen class I (HLA-I) alleles covering 12 HLA-I supertypes were predicted using the NetCTL algorithm. A subset of 179 predicted YFV and 158 predicted DENV epitopes were selected using the EpiSelect algorithm to allow for optimal coverage of viral strains. The selected predicted epitopes were synthesized and approximately 75% were found to bind the predicted restricting HLA molecule with an affinity, KD, stronger than 500 nM. The immunogenicity of 25 HLA-A*02:01, 28 HLA-A*24:02 and 28 HLA-B*07:02 binding peptides was tested in three HLA-transgenic mice models and led to the identification of 17 HLA-A*02:01, 4 HLA-A*2402 and 4 HLA-B*07:02 immunogenic peptides. The immunogenic peptides bound HLA significantly stronger than the non-immunogenic peptides. All except one of the immunogenic peptides had KD below 100 nM and the peptides with KD below 5 nM were more likely to be immunogenic. In addition, all the immunogenic peptides that were identified as having a high functional avidity had KD below 20 nM. A*02:01 transgenic mice were also inoculated twice with the 17DD YFV vaccine strain. Three of the YFV A*02:01 restricted peptides activated T-cells from the infected mice in vitro. All three peptides that elicited responses had an HLA binding affinity of 2 nM or less. The results indicate the importance of the strength of HLA binding in shaping the immune response.

Comparison of DNA vaccines producing HIV-1 Gag and LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell responses with distinct phenotypes.

Abstract Optimized DNA expression vectors encoding the native HIV-1 Gag or a fusion of Gag with the lysosomal membrane associated protein 1 (LAMP) were compared for immunogenicity upon intramuscular DNA delivery in rhesus macaques. Both vaccines elicited CD4+ T-cell responses, but with significant differences in the phenotype of the Gag-specific cells: the native Gag induced CD4+ responses with a phenotype of central memory-like T cells (CD28+ CD45RA−), whereas the LAMP/Gag chimera induced CD4+ responses with effector memory phenotype (CD28− CD45RA−). Antigen-specific T cells producing both IFN-γ and TNFα were found in the animals receiving the native Gag, whereas the LAMP/Gag chimera induced humoral responses faster. These results demonstrate that modification of intracellular Gag trafficking results in the induction of distinct immune responses. Combinations of DNA vectors encoding both forms of antigen may be more potent in eliciting anti-HIV-1 immunity.

A DNA Vaccine against Yellow Fever Virus: Development and Evaluation

Attenuated yellow fever (YF) virus 17D/17DD vaccines are the only available protection from YF infection, which remains a significant source of morbidity and mortality in the tropical areas of the world. The attenuated YF virus vaccine, which is used worldwide, generates both long-lasting neutralizing antibodies and strong T-cell responses. However, on rare occasions, this vaccine has toxic side effects that can be fatal. This study presents the design of two non-viral DNA-based antigen formulations and the characterization of their expression and immunological properties. The two antigen formulations consist of DNA encoding the full-length envelope protein (p/YFE) or the full-length envelope protein fused to the lysosomal-associated membrane protein signal, LAMP-1 (pL/YFE), aimed at diverting antigen processing/presentation through the major histocompatibility complex II precursor compartments. The immune responses triggered by these formulations were evaluated in H2b and H2d backgrounds, corresponding to the C57Bl/6 and BALB/c mice strains, respectively. Both DNA constructs were able to induce very strong T-cell responses of similar magnitude against almost all epitopes that are also generated by the YF 17DD vaccine. The pL/YFE formulation performed best overall. In addition to the T-cell response, it was also able to stimulate high titers of anti-YF neutralizing antibodies comparable to the levels elicited by the 17DD vaccine. More importantly, the pL/YFE vaccine conferred 100% protection against the YF virus in intracerebrally challenged mice. These results indicate that pL/YFE DNA is an excellent vaccine candidate and should be considered for further developmental studies.

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-γ- 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.