Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has swept the world and poses a significant global threat to lives and livelihoods, with 115 million confirmed cases and at least 2.5 million deaths from Coronavirus disease 2019 (COVID-19) in the first year of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings.Methods: We established an indirect ELISA using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n = 77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of 6 weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n = 58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva.Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and optical density (OD) values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3,200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers.Conclusion: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

41HIV-1 infection substantially increases the risk of developing tuberculosis (TB). Some 42 mechanisms, such as defects in the Th1 response to Mycobacterium tuberculosis (M.tb) in 43HIV-infected individuals have been widely reported. However, Th1-independent mechanisms 44 also contribute to protection against TB. To identify a broader spectrum of defects in TB 45 immunity during HIV infection, we examined IL-17 and IL-22 production in response to 46 mycobacterial antigens in individuals with latent TB infection (LTBI) and HIV co-infection. 47Upon stimulating with mycobacterial antigens, we observed a distinct CD4+ T helper lineage 48 producing IL-22 in the absence of IL-17 and IFN-γ. Th22 cells were present at high 49 frequencies in response to mycobacterial antigens in blood and contributed up to 50% to the 50 CD4+ T cell response to mycobacteria, comparable in magnitude to the IFN-γ Th1 response 51 (median 0.91% and 0.55%, respectively). Phenotypic characterization of Th22 cells revealed 52 that their memory differentiation was similar to M.tb-specific Th1 cells (i.e. predominantly 53 early-differentiated CD45RO+CD27+ phenotype). Moreover, CCR6 and CXCR3 expression 54 profiles of Th22 cells were similar to Th17 cells, while their CCR4 and CCR10 expression 55 patterns displayed an intermediate phenotype between Th1 and Th17 cells. Strikingly, 56 mycobacterial IL-22 responses were three-fold lower in HIV-infected individuals compared 57 to uninfected individuals, and the magnitude of responses correlated inversely with HIV viral 58 load. These data provide important insights into mycobacteria-specific T helper subsets and 59 suggest a potential role for IL-22 in protection against TB during HIV infection. Further 60 studies are needed to fully elucidate the role of IL-22 in protective TB immunity. 61 4 INTRODUCTION 62

Background: The SARS-CoV-2 pandemic has swept the world and poses a significant global threat to lives and livelihoods, with over 16 million confirmed cases and at least 650 000 deaths from COVID-19 in the first 7 months of the pandemic. Developing tools to measure seroprevalence and understand protective immunity to SARS-CoV-2 is a priority. We aimed to develop a serological assay using plant-derived recombinant viral proteins, which represent important tools in less-resourced settings. Methods: We established an indirect enzyme-linked immunosorbent assay (ELISA) using the S1 and receptor-binding domain (RBD) portions of the spike protein from SARS-CoV-2, expressed in Nicotiana benthamiana. We measured antibody responses in sera from South African patients (n=77) who had tested positive by PCR for SARS-CoV-2. Samples were taken a median of six weeks after the diagnosis, and the majority of participants had mild and moderate COVID-19 disease. In addition, we tested the reactivity of pre-pandemic plasma (n=58) and compared the performance of our in-house ELISA with a commercial assay. We also determined whether our assay could detect SARS-CoV-2-specific IgG and IgA in saliva. Results: We demonstrate that SARS-CoV-2-specific immunoglobulins are readily detectable using recombinant plant-derived viral proteins, in patients who tested positive for SARS-CoV-2 by PCR. Reactivity to S1 and RBD was detected in 51 (66%) and 48 (62%) of participants, respectively. Notably, we detected 100% of samples identified as having S1-specific antibodies by a validated, high sensitivity commercial ELISA, and OD values were strongly and significantly correlated between the two assays. For the pre-pandemic plasma, 1/58 (1.7%) of samples were positive, indicating a high specificity for SARS-CoV-2 in our ELISA. SARS-CoV-2-specific IgG correlated significantly with IgA and IgM responses. Endpoint titers of S1- and RBD-specific immunoglobulins ranged from 1:50 to 1:3200. S1-specific IgG and IgA were found in saliva samples from convalescent volunteers. Conclusions: We demonstrate that recombinant SARS-CoV-2 proteins produced in plants enable robust detection of SARS-CoV-2 humoral responses. This assay can be used for seroepidemiological studies and to measure the strength and durability of antibody responses to SARS-CoV-2 in infected patients in our setting.

HIV-1 infection substantially increases the risk of developing tuberculosis (TB). Mechanisms such as defects in the Th1 response to Mycobacterium tuberculosis in HIV-infected persons have been widely reported. However, Th1-independent mechanisms also contribute to protection against TB. To identify a broader spectrum of defects in TB immunity during HIV infection, we examined IL-17A and IL-22 production in response to mycobacterial Ags in peripheral blood of persons with latent TB infection and HIV coinfection. Upon stimulating with mycobacterial Ags, we observed a distinct CD4 + Th lineage producing IL-22 in the absence of IL-17A and IFN-g. Mycobacteria-specific Th22 cells were present at high frequencies in blood and contributed up to 50% to the CD4 + T cell response to mycobacteria, comparable in magnitude to the IFN-g Th1 response (median 0.91% and 0.55%, respectively). Phenotypic characterization of Th22 cells revealed that their memory differentiation was similar to M. tuberculosisspecific Th1 cells (i.e., predominantly early differentiated CD45RO + CD27 + phenotype). Moreover, CCR6 and CXCR3 expression profiles of Th22 cells were similar to Th17 cells, whereas their CCR4 and CCR10 expression patterns displayed an intermediate phenotype between Th1 and Th17 cells. Strikingly, mycobacterial IL-22 responses were 3-fold lower in HIV-infected persons compared with uninfected persons, and the magnitude of responses correlated inversely with HIV viral load. These data provide important insights into mycobacteria-specific Th subsets in humans and suggest a potential role for IL-22 in protection against TB during HIV infection. Further studies are needed to fully elucidate the role of IL-22 in protective TB immunity.

The development of a highly effective tuberculosis (TB) vaccine is likely dependent on our understanding of what constitutes a protective immune response to TB. Accumulating evidence suggests that CD4 + T cells producing IL-22, a distinct subset termed "Th22" cells, may contribute to protective immunity to TB. Thus, we characterized Mycobacterium tuberculosisspecific Th22 (and Th1 and Th17) cells in 72 people with latent TB infection or TB disease, with and without HIV-1 infection. We investigated the functional properties (IFN-g, IL-22, and IL-17 production), memory differentiation (CD45RA, CD27, and CCR7), and activation profile (HLA-DR) of M. tuberculosisspecific CD4 + T cells. In HIV-uninfected individuals with latent TB infection, we detected abundant circulating IFN-gproducing CD4 + T cells (median, 0.93%) and IL-22producing CD4 + T cells (median, 0.46%) in response to M. tuberculosis. The frequency of IL-17producing CD4 + T cells was much lower, at a median of 0.06%. Consistent with previous studies, IL-22 was produced by a distinct subset of CD4 + T cells and not coexpressed with IL-17. M. tuberculosisspecific IL-22 responses were markedly reduced (median, 0.08%) in individuals with TB disease and HIV coinfection compared with IFN-g responses. M. tuberculosisspecific Th22 cells exhibited a distinct memory and activation phenotype compared with Th1 and Th17 cells. Furthermore, M. tuberculosisspecific IL-22 was produced by conventional CD4 + T cells that required TCR engagement. In conclusion, we confirm that Th22 cells are a component of the human immune response to TB. Depletion of M. tuberculosisspecific Th22 cells during HIV coinfection may contribute to increased risk of TB disease.