A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre 'synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved

AstraZeneca COVID-19 vaccine induces robust broadly cross-reactive antibody responses in Malawian adults previously infected with SARS-CoV-2

Background: Binding and neutralising anti-Spike antibodies play a key role in immune defence against SARS-CoV-2 infection. Since it is known that antibodies wane with time and new immune-evasive variants are emerging, we aimed to assess the dynamics of anti-Spike antibodies in an African adult population with prior SARS-CoV-2 infection and to determine the effect of subsequent COVID-19 vaccination. Methods: Using a prospective cohort design, we recruited adults with prior laboratory-confirmed mild/moderate COVID-19 in Blantyre, Malawi, and followed them up for 270 days (n = 52). A subset of whom subsequently received a single dose of the AstraZeneca COVID-19 vaccine (ChAdOx nCov-19) (n = 12). We measured the serum concentrations of anti-Spike and receptor-binding domain (RBD) IgG antibodies using a Luminex-based assay. Anti-RBD antibody cross-reactivity across SARS-CoV-2 variants of concern (VOC) was measured using a haemagglutination test. A pseudovirus neutralisation assay was used to measure neutralisation titres across VOCs. Ordinary or repeated measures one-way ANOVA was used to compare log10 transformed data, with p value adjusted for multiple comparison using Šídák's or Holm-Šídák's test. Results: We show that neutralising antibodies wane within 6 months post mild/moderate SARS-CoV-2 infection (30–60 days vs. 210–270 days; Log ID50 6.8 vs. 5.3, p = 0.0093). High levels of binding anti-Spike or anti-RBD antibodies in convalescent serum were associated with potent neutralisation activity against the homologous infecting strain (p < 0.0001). A single dose of the AstraZeneca COVID-19 vaccine following mild/moderate SARS-CoV-2 infection induced a 2 to 3-fold increase in anti-Spike and -RBD IgG levels 30 days post-vaccination (both, p < 0.0001). The anti-RBD IgG antibodies from these vaccinated individuals were broadly cross-reactive against multiple VOCs and had neutralisation potency against original D614G, beta, and delta variants. Conclusions: These findings show that the AstraZeneca COVID-19 vaccine is an effective booster for waning cross-variant antibody immunity after initial priming with SARS-CoV-2 infection. The potency of hybrid immunity and its potential to maximise the benefits of COVID-19 vaccines needs to be taken into consideration when formulating vaccination policies in sub-Saharan Africa, where there is still limited access to vaccine doses

Asymptomatic HIV-infected Individuals on Antiretroviral Therapy Exhibit Impaired Lung CD4+T-Cell Responses to Mycobacteria

Rationale: HIV-infected persons on antiretroviral therapy (ART) remain at higher risk of pulmonary tuberculosis (TB) than HIV-uninfected individuals. This increased susceptibility may be caused by impairment of alveolar macrophage (AM) function and/or mycobacteria-specific alveolar CD4+ T-cell responses observed in HIV-infected ART-naive adults. Objectives: To determine whether ART was associated with improvement in both AM function, assessed by phagosomal proteolysis, and alveolar CD4+ T-cell responses to Mycobacterium in HIV-infected individuals. Methods: Peripheral blood was drawn and bronchoalveolar lavage (BAL) performed on healthy, 35 HIV-uninfected, 25 HIV-infected ART-naive, and 50 HIV-infected ART-treated asymptomatic adults. Phagosomal proteolysis of AM was assessed with fluorogenic beads. Mycobacteria-specific CD4+ T-cell responses were measured by intracellular cytokine staining. Measurements and Main Results: HIV-infected adults on ART exhibited lower plasma HIV viral load and higher blood CD4+ T-cell count than ART-naive adults. AM proteolysis and total mycobacteria-specific Th1 CD4+ T-cell responses in individuals on ART for greater than or equal to 4 years were similar to HIV-uninfected control subjects but those on ART for less than 4 years had impaired responses. Total influenza-specific alveolar Th1 CD4+ T-cell responses were intact in all individuals receiving ART. In contrast, BAL and blood mycobacteria-specific polyfunctional CD4+ T-cell responses were impaired in adults on ART irrespective of duration. Conclusions: AM and mycobacteria-specific alveolar CD4+ T-cell responses in HIV-infected adults on ART for less than 4 years are impaired and may partly explain the high risk of TB in HIV-infected individuals on ART. Strategies to augment ART to improve lung immune cell function and reduce the high incidence of TB in HIV-infected adults who initiate ART should be investigated

Asymptomatic HIV-infected Individuals on Antiretroviral Therapy Exhibit Impaired Lung CD4 +

Rationale: HIV-infected persons on antiretroviral therapy (ART) remain at higher risk of pulmonary tuberculosis (TB) than HIV-uninfected individuals. This increased susceptibility may be caused by impairment of alveolar macrophage (AM) function and/or mycobacteria-specific alveolar CD4+ T-cell responses observed in HIV-infected ART-naive adults. Objectives: To determine whether ART was associated with improvement in both AM function, assessed by phagosomal proteolysis, and alveolar CD4+ T-cell responses to Mycobacterium in HIV-infected individuals. Methods: Peripheral blood was drawn and bronchoalveolar lavage (BAL) performed on healthy, 35 HIV-uninfected, 25 HIV-infected ART-naive, and 50 HIV-infected ART-treated asymptomatic adults. Phagosomal proteolysis of AM was assessed with fluorogenic beads. Mycobacteria-specific CD4+ T-cell responses were measured by intracellular cytokine staining. Measurements and Main Results: HIV-infected adults on ART exhibited lower plasma HIV viral load and higher blood CD4+ T-cell count than ART-naive adults. AM proteolysis and total mycobacteria-specific Th1 CD4+ T-cell responses in individuals on ART for greater than or equal to 4 years were similar to HIV-uninfected control subjects but those on ART for less than 4 years had impaired responses. Total influenza-specific alveolar Th1 CD4+ T-cell responses were intact in all individuals receiving ART. In contrast, BAL and blood mycobacteria-specific polyfunctional CD4+ T-cell responses were impaired in adults on ART irrespective of duration. Conclusions: AM and mycobacteria-specific alveolar CD4+ T-cell responses in HIV-infected adults on ART for less than 4 years are impaired and may partly explain the high risk of TB in HIV-infected individuals on ART. Strategies to augment ART to improve lung immune cell function and reduce the high incidence of TB in HIV-infected adults who initiate ART should be investigated