Expanding the Malaria Antibody Toolkit: Development and Characterisation of Plasmodium falciparum RH5, CyRPA, and CSP Recombinant Human Monoclonal Antibodies.

Malaria, an infection caused by apicomplexan parasites of the genus Plasmodium, continues to exact a significant toll on public health with over 200 million cases world-wide, and annual deaths in excess of 600,000. Considerable progress has been made to reduce malaria burden in endemic countries in the last two decades. However, parasite and mosquito resistance to frontline chemotherapies and insecticides, respectively, highlights the continuing need for the development of safe and effective vaccines. Here we describe the development of recombinant human antibodies to three target proteins from Plasmodium falciparum: reticulocyte binding protein homologue 5 (PfRH5), cysteine-rich protective antigen (PfCyRPA), and circumsporozoite protein (PfCSP). All three proteins are key targets in the development of vaccines for blood-stage or pre-erythrocytic stage infections. We have developed potent anti-PfRH5, PfCyRPA and PfCSP monoclonal antibodies that will prove useful tools for the standardisation of assays in preclinical research and the assessment of these antigens in clinical trials. We have generated some very potent anti-PfRH5 and anti-PfCyRPA antibodies with some clones >200 times more potent than the polyclonal anti-AMA-1 antibodies used for the evaluation of blood stage antigens. While the monoclonal and polyclonal antibodies are not directly comparable, the data provide evidence that these new antibodies are very good at blocking invasion. These antibodies will therefore provide a valuable resource and have potential as biological standards to help harmonise pre-clinical malaria research.

Efficacy and immunogenicity of different BCG doses in BALB/c and CB6F1 mice when challenged with H37Rv or Beijing HN878.

Two strains of mice (BALB/c and CB6F1) were vaccinated with a range of Bacille Calmette-Guérin (BCG) Danish doses from 3 × 105 to 30 CFU/mouse, followed by aerosol infection with Mtb (H37Rv or West-Beijing HN878 strain). The results indicated that both strains of mice when infected with HN878 exhibited significant protection in their lungs with BCG doses at 3 × 105-3000 CFU (BALB/c) and 3 × 105-300 CFU (CB6F1). Whereas, a significant protection was seen in both strains of mice with BCG doses at 3 × 105-300 CFU when infected with H37Rv. A significant increase in the frequencies of BCG-specific IFNγ+ IL2+ TNFα+ CD4 T cells in the BCG doses at 3 × 105-3000 CFU (BALB/c) and 3 × 105-300 CFU (CB6F1) was seen. The IFNγ+ IL2+ TNFα+ CD4 T cells correlated with the Mtb burden in the lungs of HN878 infected mice (BALB/c and CB6F1) whereas, IFNγ+ TNFα+ CD4 T cells correlated with the BALB/c mice infected with H37Rv or HN878. The BCG dose at 3000 CFU (an equivalent single human dose in the mice by body weight) is protective in both strains of mice infected with H37Rv or HN878 and may serve an interesting dose to test new TB vaccine in a preclinical animal model.

Comparative fitness analysis of D-cycloserine resistant mutants reveals both fitness-neutral and high-fitness cost genotypes.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations.