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

Development and pre-clinical evaluation of HIV-1 vaccines

Infants born to HIV-1 positive mothers are at risk of acquiring the infection through pro-longed breastfeeding due to the presence of HIV-1 cell-free RNA and cell-associated DNA in breast milk. However, there is limited focus on vaccines to prevent mother-to-child transmission (MTCT) via breastfeeding. Mycobacterium tuberculosis (M. tuberculosis) the causative agent of Tuberculosis (TB) is the most common cause of AIDS-related deaths. Most infants in Africa receive Bacillus Calmette-Guérin (BCG) at birth or soon after birth and it is the only licensed vaccine for TB. The development of a dual platform vaccine against HIV-1 and TB would be a logical effort to combat these two deadly diseases. Thus, rBCG expressing an HIV-1 derived immunogen may induce HIV-1 responses at birth and these responses can be boosted at adolescence, by a heterologous vector such as modified vaccinia Ankara (MVA) or Chimpanzee adenovirus serotype 63 (ChAdV63). In the first study, I assessed BCG-based vaccines derived from BCG Danish SSI-1331 (BCG1331), expressing an HIV-1 immunogen HIVconsv either by an episomal plasmid (BCG.HIVconsv401epi) or integrated into the BCG chromosome (BCG.HIVconsv401int) in a prime-boost regimen. BALB/c mice were immunised with the different prime-boost regimens. rBCG alone was unable to induce detectable HIV-1-specific T-cell responses, however, when used in a prime-boost strategy, elevated HIV-1-specific T-cell responses were observed. In the second study, I aimed to construct marker-less mycobacterium-vectored HIV-1 vaccines using the operator-repressor titration (ORT®) system as an alternative system for antibiotic resistance gene free vaccines. This rBCG vaccine would express the HIVconsv immunogen. I first constructed plasmids carrying the lac operator lacO and tetracycline operator tetO to enable use in ORT Escherichia coli (E.coli) and Mycobacterium strains, respectively. The ORT system was successful in E. coli and not in mycobacterium. I also constructed plasmids carrying mycobacterium essential genes that would allow for genetic manipulation in Mycobacterium and the use of ORT in mycobacterium. Although the plasmid construction was successful, in the end, genetic manipulations in Mycobacterium and the production of an ORT based BCG (ORT-VAC) was not successful. Finally, I evaluated the immunogenicity of conventional DNA plasmid pTH.HIVconsv compared to Semliki Forest virus replicon DREP.HIVconsv in rhesus macaques. Immunisations were done in a prime-boost strategy with heterologous vectors MVA or ChAdV63 delivering the same immunogen, HIVconsv. It was found that DREP.HIVconsv which was at least 20-fold lower dose than pTH.HIVconsv was capable of inducing comparable T-cell responses and in some experiments, the responses were superior to the conventional DNA plasmid pTH.HIVconsv.</p

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

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

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

High SARS-CoV-2 seroprevalence in health care workers but relatively low numbers of deaths in urban Malawi

Background: In low-income countries, like Malawi, important public health measures including social distancing or a lockdown have been challenging to implement owing to socioeconomic constraints, leading to predictions that the COVID-19 pandemic would progress rapidly. However, due to limited capacity to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, there are no reliable estimates of the true burden of infection and death.  We, therefore, conducted a SARS-CoV-2 serosurvey amongst health care workers (HCWs) in Blantyre city to estimate the cumulative incidence of SARS-CoV-2 infection in urban Malawi. Methods: We recruited 500 otherwise asymptomatic HCWs from Blantyre City (Malawi) from 22 nd May 2020 to 19 th June 2020 and serum samples were collected from all participants. A commercial ELISA was used to measure SARS-CoV-2 IgG antibodies in serum. Results: A total of 84 participants tested positive for SARS-CoV-2 antibodies. The HCWs with positive SARS-CoV-2 antibody results came from different parts of the city. The adjusted seroprevalence of SARS-CoV-2 antibodies was 12.3% [CI 8.2 - 16.5]. Using age-stratified infection fatality estimates reported from elsewhere, we found that at the observed adjusted seroprevalence, the number of predicted deaths was eight times the number of reported deaths. Conclusions: The high seroprevalence of SARS-CoV-2 antibodies among HCWs and the discrepancy in the predicted versus reported deaths suggests that there was early exposure but slow progression of COVID-19 epidemic in urban Malawi. This highlights the urgent need for development of locally parameterised mathematical models to more accurately predict the trajectory of the epidemic in sub-Saharan Africa for better evidence-based policy decisions and public health response planning