Pre-clinical development of BCG.HIVA(CAT), an antibiotic-free selection strain, for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVACAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVACAT. All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVACAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVACAT vaccine strain. The BCG.HIVACAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVACAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8+ T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVACAT-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth

T cell phenotypes in COVID-19 - a living review

COVID-19 is characterized by profound lymphopenia in the peripheral blood, and the remaining T cells display altered phenotypes, characterized by a spectrum of activation and exhaustion. However, antigen-specific T cell responses are emerging as a crucial mechanism for both clearance of the virus and as the most likely route to long-lasting immune memory that would protect against re-infection. Therefore, T cell responses are also of considerable interest in vaccine development. Furthermore, persistent alterations in T cell subset composition and function post-infection have important implications for patients’ long-term immune function. In this review, we examine T cell phenotypes, including those of innate T cells, in both peripheral blood and lungs, and consider how key markers of activation and exhaustion correlate with, and may be able to predict, disease severity. We focus on SARS-CoV-2-specific T cells to elucidate markers that may indicate formation of antigen-specific T cell memory. We also examine peripheral T cell phenotypes in recovery and the likelihood of long-lasting immune disruption. Finally, we discuss T cell phenotypes in the lung as important drivers of both virus clearance and tissue damage. As our knowledge of the adaptive immune response to COVID-19 rapidly evolves, it has become clear that while some areas of the T cell response have been investigated in some detail, others, such as the T cell response in children remain largely unexplored. Therefore, this review will also highlight areas where T cell phenotypes require urgent characterisation

Pre-clinical development of BCG.HIVA(CAT), an antibiotic-free selection strain, for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG

In the past, we proposed to develop a heterologous recombinant BCG prime-recombinant modified vaccinia virus Ankara (MVA) boost dual pediatric vaccine platform against transmission of breast milk HIV-1 and Mycobacterium tuberculosis (Mtb). In this study, we assembled an E. coli-mycobacterial shuttle plasmid pJH222.HIVACAT expressing HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism based on Operator-Repressor Titration (ORT) system for plasmid selection and maintenance in E. coli and lysine complementation in mycobacteria. This shuttle plasmid was electroporated into parental lysine auxotroph (safer) strain of BCG to generate vaccine BCG.HIVACAT. All procedures complied with Good Laboratory Practices (GLPs). We demonstrated that the episomal plasmid pJH222.HIVACAT was stable in vivo over a 20-week period, and genetically and phenotypically characterized the BCG.HIVACAT vaccine strain. The BCG.HIVACAT vaccine in combination with MVA.HIVA induced HIV-1- and Mtb-specific interferon γ-producing T-cell responses in newborn and adult BALB/c mice. On the other hand, when adult mice were primed with BCG.HIVACAT and boosted with MVA.HIVA.85A, HIV-1-specific CD8+ T-cells producing IFN-γ, TNF-α, IL-2 and CD107a were induced. To assess the biosafety profile of BCG.HIVACAT-MVA.HIVA regimen, body mass loss of newborn mice was monitored regularly throughout the vaccination experiment and no difference was observed between the vaccinated and naïve groups of animals. Thus, we demonstrated T-cell immunogenicity of a novel, safer, GLP-compatible BCG-vectored vaccine using prototype immunogen HIVA. Second generation immunogens derived from HIV-1 as well as other major pediatric pathogens can be constructed in a similar fashion to prime protective responses soon after birth

Phenotypic characterization of the BCG.HIVA^CAT.

<p>We assessed the phenotype of lysine auxotrophy, lysine complementation and kanamycin resistance of BCG.HIVA<b>^CAT</b> strain. (<b>A</b>) BCG lysine auxotroph strain plated on non-lysine supplemented 7H10; (<b>B</b>) BCG lysine auxotroph strain plated on lysine supplemented 7H10; (<b>C</b>) BCG.HIVA<b>^CAT</b> plated on 7H10 without lysine and kanamycin supplementation; (<b>D</b>) BCG.HIVA^CAT plated on 7H10 without lysine supplementation and with kanamycin.</p

Induction of HIV-1- and <i>Mtb</i>-specific T-cells responses by the BCG.HIVA^CAT prime - MVA.HIVA boost regimen.

<p>(<b>A</b>) Adult mice (7-weeks-old) immunized with either 10⁴ or 10⁵ cfu of BCG.HIVA^CAT alone (subcutaneously), 10⁶ pfu of MVA.HIVA.85A alone (intramuscularly), or 10⁴ or 10⁵ cfu of BCG.HIVA^CAT as a prime and boosted with 10⁶ pfu of MVA.HIVA.85A (left to right). Mice were sacrificed 2 weeks later for T-cell analysis. (<b>B</b>) Analysis of IFN-γ, TNF-α, CD107a and IL-2 vaccine elicited HIV-1-specific CD8⁺ T-cell responses. The frequencies of cells producing cytokine are shown. Data are presented as group medians as well as individual animal responses (n = 5). (<b>C</b>) Adult and newborn mice (7-days-old) were either left unimmunized or immunized with 2×10⁶ cfu of BCG.HIVA^CAT (intradermal and subcutaneous route respectively) and subsequently given a booster dose of 10⁶ pfu of MVA.HIVA (intramuscularly) at 14 weeks post BCG immunization, and sacrificed 3 weeks later. (<b>D</b>) Analysis of IFN-γ vaccine elicited HIV-1-specific CD8⁺ T-cell responses. The frequencies of cells producing cytokine are shown. Data are presented as group medians as well as individual animal responses (n = 4). (<b>E</b>) PPD-specific T-cell responses elicited by BCG.HIVA^CAT. Immune responses to BCG were assessed in an <i>ex vivo</i> IFN-γ ELISPOT assay using PPD as the antigen. The median spot-forming units (SFU) per 10⁶ splenocytes for each group of mice (n = 4) as well as individual animal responses is shown. * = p<0.05.</p

Construction of the BCG.HIVA^CAT vaccine strain.

<p>(<b>A</b>) A synthetic GC-rich HIVA gene was fused to the region encoding the 19-kDa lipoprotein signal sequence and inserted into the episomal pJH222 <i>E. coli</i>-mycobacterium shuttle plasmid. This plasmid contains kanamycin resistance (<i>aph</i>) and complementing <i>lysA</i> genes and an <i>E. coli</i> origin of replication (oriE). In addition, pJH222 contained the mycobacterial origin of replication (oriM). The BALB/c mouse T-cell and MAb Pk epitopes used in this work are depicted. P α-Ag, <i>M. tuberculosis</i> α-antigen promoter; P<i>HSP60</i>, heat shock protein 60 gene promoter. The <i>aph</i> gene was removed by SpeI digestion and the <i>lacO</i> sequence was inserted and transformed into <i>E. coli</i> DH1<i>lacdapD</i> strain. (<b>B</b>) Immunodot of BCG.HIVA^CAT lysates. Dot 1: BCG wild type (negative control); Dot 2, 3, 4 and 5: clone 3, clone 7, clone 9 and clone 10 of BCG.HIVA^CAT; Dot 6: BCG.HIVA²²² (positive control). HIVA peptide was detected using the anti-Pk MAb followed by horseradish peroxidase-Goat-anti-Mouse and enhanced chemiluminescence (ECL) detection. (<b>C</b>) <i>In vivo</i> plasmid stability of BCG.HIVA^CAT harboring pJH222.HIVA^CAT. Mice were injected s.c. with 10⁵ cfu of BCG.HIVA^CAT and boosted i.m. with 10⁶ pfu of MVA.HIVA, spleens were homogenized 20 weeks after BCG inoculation and the recovered rBCG colonies were tested for the presence of the HIVA DNA coding sequence by PCR. Lanes 1 to 6: Six rBCG colonies were recovered in the non-lysine supplemented plate; lane 7: Molecular weight marker; lane 8: Plasmid DNA positive control; lane 9: Distilled water (negative control).</p

BCG.HIVA^CAT prime and MVA.HIVA boost safety in newborn mice.

<p>(<b>A</b>) Newborn mice were either left unimmunized or immunized with 2×10⁶ cfu of BCG wild type, BCG:HIVA²²² or BCG.HIVA^CAT by subcutaneous route and subsequently given a booster dose of 10⁶ pfu of MVA.HIVA at week 14. (<b>B</b>) The body weight was weekly recorded, and the mean for each group of mice is shown (n = 10). Data from naive mice are presented as mean ± 2 SEM (n = 6); At specific time points the weight differences between vaccinated and naïve mice group were analyzed by ANOVA test (arrowheads).</p