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. 2022 Aug 3;10(8):1246.
doi: 10.3390/vaccines10081246.

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

Franciane Mouradian Emidio Teixeira  1   2 , Luana de Mendonça Oliveira  1   2 , Anna Julia Pietrobon  1   2 , Érika Machado de Salles  2 , Maria Regina D'Império Lima  2 , Isabelle Freire Tabosa Viana  3 , Roberto Dias Lins  3 , Paula Ordonhez Rigato  4 , Ernesto Torres de Azevedo Marques  3   5 , Alberto José da Silva Duarte  1 , Maria Notomi Sato  1   2
Affiliations

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

Franciane Mouradian Emidio Teixeira et al. Vaccines (Basel). .

Abstract

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.

Keywords: DNA vaccine; HIV; germinal center; immunogenicity; neonate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Generation of Gag-specific cellular response by neonatal C57BL/6 mouse immunization. Neonate C57BL/6 mice were immunized (ID) with 2 doses of LAMP/Gag or Gag DNA vaccines at a 25-day interval: (A) Gag-specific IFN-γ-secreting cells were evaluated 10 days after boost by ELISPOT assay in response to class I (361–411aa), class II (121–171aa and 281–331aa), and class I plus II immunodominant peptide pools or p24 recombinant protein; (B) The cellular response was evaluated, as shown in (A) 4 months after immunization. Saline was used as a negative control (n = 6). Assays were performed in duplicate, and the results were subtracted from the baseline value. The data are shown as medians. * p ≤ 0.05; ** p ≤ 0.01.
Figure 2
Figure 2
Gag-specific humoral response and plasma-cell generation. Neonate mice were immunized with Gag or LAMP/Gag vaccines: (A) Anti-p24 (HIV-1) IgG antibodies were detected by ELISA in the serum obtained from immunized mice within 10 days after the boost (Saline group: n = 4); (B) Anti-Gag IgG antibody binding curves to the HIV-1 p24 protein were measured by microscale thermophoresis, where serum binding curves acquired from 2 selected mouse-serum samples were plotted in terms of fraction bound (%) vs. concentration of total IgG; (C) The number of anti-p24 IgG1 ASCs was assessed in the spleen by ELISPOT for B cells in the short-term (30 days) and long-term (4 months) after immunization (Saline group: n = 3–6); (D) The number of ASCs was also evaluated in the bone marrow at 4 months. The data are shown as medians. * p ≤ 0.05; ** p ≤ 0.01.
Figure 3
Figure 3
TFH cell generation in draining inguinal lymph nodes. Neonate mice were immunized with LAMP/Gag or Gag DNA vaccines, and the analysis was performed in ILNs within 3 or 7 days after the boost: (A) Frequency of TFH cells (CD4 + CXCR5 + PD-1 + Bcl6 +) in ILNs by flow cytometry (Saline group: n = 5); (B,C) IL-21, Bcl-6, and BLIMP-1 mRNA expression evaluated in ILNs by real-time PCR at 3 and 7 days after boost, respectively.(Saline group: n = 3–6). The data are shown as medians.
Figure 4
Figure 4
GC site formation and antibody-binding affinity: (A) The frequency of GC B cells (CD19 + CD95 + GL-7+) was evaluated within 3 days after the boost in neonatal immunization in ILNs by flow cytometry (Saline group: n = 8); (B) mRNA expression of genes for AID enzyme by real-time PCR of cells obtained from ILNs 3 days post-boost (Saline group: n = 3); (C) Representative ILN sections from immunized neonates (n = 3/group) were obtained within 3 days after the boost, and GC was identified by immunofluorescence (CD3: red; GL-7: green; original magnification ×10); (D) Representative expression of AID in ILN tissues by immunofluorescence as described in B (CD19: red; AID: green; original magnification ×10). ** p ≤ 0,01; *** p ≤ 0001.
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