Optimisation and Validation of a conventional ELISA and cut-offs for detecting and quantifying anti-SARS-CoV-2 Spike, RBD, and Nucleoprotein IgG, IgM, and IgA antibodies in Uganda.

There is an urgent need for better immunoassays to measure antibody responses as part of immune-surveillance activities and to profile immunological responses to emerging SARS-CoV-2 variants. We optimised and validated an in-house conventional ELISA to identify and quantify SARS-CoV-2 spike- (S-), receptor binding domain- (RBD-), and nucleoprotein- (N-) directed IgG, IgM, and IgA binding antibodies in the Ugandan population and similar settings. Pre- and post-pandemic specimens were used to compare the utility of mean ± 2SD, mean ± 3SD, 4-fold above blanks, bootstrapping, and receiver operating characteristic (ROC) analyses in determining optimal cut-off optical densities at 450 nm (OD) for discriminating between antibody positives and negatives. "Limits of detection" (LOD) and "limits of quantitation" (LOQ) were validated alongside the assay's uniformity, accuracy, inter-assay and inter-operator precision, and parallelism. With spike-directed sensitivity and specificity of 95.33 and 94.15%, respectively, and nucleoprotein sensitivity and specificity of 82.69 and 79.71%, ROC was chosen as the best method for determining cutoffs. Accuracy measurements were within the expected CV range of 25%. Serum and plasma OD values were highly correlated (r = 0.93, p=0.0001). ROC-derived cut-offs for S-, RBD-, and N-directed IgG, IgM, and IgA were 0.432, 0.356, 0.201 (S), 0.214, 0.350, 0.303 (RBD), and 0.395, 0.229, 0.188 (N). The sensitivity and specificity of the S-IgG cut-off were equivalent to the WHO 20/B770-02 S-IgG reference standard at 100% level. Spike negative IgG, IgM, and IgA ODs corresponded to median antibody concentrations of 1.49, 3.16, and 0 BAU/mL, respectively, consistent with WHO low titre estimates. Anti-spike IgG, IgM, and IgA cut-offs were equivalent to 18.94, 20.06, and 55.08 BAU/mL. For the first time, we provide validated parameters and cut-off criteria for the in-house detection of subclinical SARS-CoV-2 infection and vaccine-elicited binding antibodies in the context of Sub-Saharan Africa and populations with comparable risk factors

Rapid, early, and potent Spike-directed IgG, IgM, and IgA distinguish asymptomatic from mildly symptomatic COVID-19 in Uganda, with IgG persisting for 28 months.

INTRODUCTION: Understanding how spike (S)-, nucleoprotein (N)-, and RBD-directed antibody responses evolved in mild and asymptomatic COVID-19 in Africa and their interactions with SARS-CoV-2 might inform development of targeted treatments and vaccines. METHODS: Here, we used a validated indirect in-house ELISA to characterise development and persistence of S- and N-directed IgG, IgM, and IgA antibody responses for 2430 SARS-CoV-2 rt-PCR-diagnosed Ugandan specimens from 320 mild and asymptomatic COVID-19 cases, 50 uninfected contacts, and 54 uninfected non-contacts collected weekly for one month, then monthly for 28 months. RESULTS: During acute infection, asymptomatic patients mounted a faster and more robust spike-directed IgG, IgM, and IgA response than those with mild symptoms (Wilcoxon rank test, p-values 0.046, 0.053, and 0.057); this was more pronounced in males than females. Spike IgG antibodies peaked between 25 and 37 days (86.46; IQR 29.47-242.56 BAU/ml), were significantly higher and more durable than N- and RBD IgG antibodies and lasted for 28 months. Anti-spike seroconversion rates consistently exceeded RBD and nucleoprotein rates. Spike- and RBD-directed IgG antibodies were positively correlated until 14 months (Spearman's rank correlation test, p-values 0.0001 to 0.05), although RBD diminished faster. Significant anti-spike immunity persisted without RBD. 64% and 59% of PCR-negative, non-infected non-contacts and suspects, exhibited baseline SARS-CoV-2 N-IgM serological cross-reactivity, suggesting undetected exposure or abortive infection. N-IgG levels waned after 787 days, while N-IgM levels remained undetectable throughout. DISCUSSION: Lower N-IgG seroconversion rates and the absence of N-IgM indicate that these markers substantially underestimate the prior exposure rates. Our findings provide insights into the development of S-directed antibody responses in mild and asymptomatic infections, with varying degrees of symptoms eliciting distinct immune responses, suggesting distinct pathogenic pathways. These longer-lasting data inform vaccine design, boosting strategies, and surveillance efforts in this and comparable settings

HIV-1 Gag-Pol Sequences from Ugandan Early Infections Reveal Sequence Variants Associated with Elevated Replication Capacity.

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication

Insulin-like growth factor (IGF)-I obliterates the pregnancy-associated protection against mammary carcinogenesis in rats: evidence that IGF-I enhances cancer progression through estrogen receptor-α activation via the mitogen-activated protein kinase pathway

INTRODUCTION: Pregnancy protects against breast cancer development in humans and rats. Parous rats have persistently reduced circulating levels of growth hormone, which may affect the activity of the growth hormone/insulin-like growth factor (IGF)-I axis. We investigated the effects of IGF-I on parity-associated protection against mammary cancer. METHODS: Three groups of rats were evaluated in the present study: IGF-I-treated parous rats; parous rats that did not receive IGF-I treatment; and age-matched virgin animals, which also did not receive IGF-I treatment. Approximately 60 days after N-methyl-N-nitrosourea injection, IGF-I treatment was discontinued and all of the animal groups were implanted with a silastic capsule containing 17β-estradiol and progesterone. The 17β-estradiol plus progesterone treatment continued for 135 days, after which the animals were killed. RESULTS: IGF-I treatment of parous rats increased mammary tumor incidence to 83%, as compared with 16% in parous rats treated with 17β-estradiol plus progesterone only. Tumor incidence and average number of tumors per animal did not differ between IGF-I-treated parous rats and age-matched virgin rats. At the time of N-methyl-N-nitrosourea exposure, DNA content was lowest but the α-lactalbumin concentration highest in the mammary glands of untreated parous rats in comparison with age-matched virgin and IGF-I-treated parous rats. The protein levels of estrogen receptor-α in the mammary gland was significantly higher in the age-matched virgin animals than in untreated parous and IGF-I-treated parous rats. Phosphorylation (activation) of the extracellular signal-regulated kinase-1/2 (ERK1/2) and expression of the progesterone receptor were both increased in IGF-I-treated parous rats, as compared with those in untreated parous and age-matched virgin rats. Expressions of cyclin D(1 )and transforming growth factor-β(3 )in the mammary gland were lower in the age-matched virgin rats than in the untreated parous and IGF-I-treated parous rats. CONCLUSION: We argue that tumor initiation (transformation and fixation of mutations) may be similar in parous and age-matched virgin animals, suggesting that the main differences in tumor formation lie in differences in tumor progression caused by the altered hormonal environment associated with parity. Furthermore, we provide evidence supporting the notion that tumor growth promotion seen in IGF-I-treated parous rats is caused by activation of estrogen receptor-α via the Raf/Ras/mitogen-activated protein kinase cascade

Improving Salmonella vector with rec mutation to stabilize the DNA cargoes

<p>Abstract</p> <p>Background</p> <p><it>Salmonella </it>has been employed to deliver therapeutic molecules against cancer and infectious diseases. As the carrier for target gene(s), the cargo plasmid should be stable in the bacterial vector. Plasmid recombination has been reduced in <it>E. coli </it>by mutating several genes including the <it>recA</it>, <it>recE</it>, <it>recF </it>and <it>recJ</it>. However, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in <it>Salmonella enterica</it>.</p> <p>Results</p> <p>The effect of <it>recA</it>, <it>recF </it>and <it>recJ </it>deletions on DNA recombination was examined in three serotypes of <it>Salmonella enterica</it>. We found that (1) intraplasmid recombination between direct duplications was RecF-independent in Typhimurium and Paratyphi A, but could be significantly reduced in Typhi by a Δ<it>recA </it>or Δ<it>recF </it>mutation; (2) in all three <it>Salmonella </it>serotypes, both Δ<it>recA </it>and Δ<it>recF </it>mutations reduced intraplasmid recombination when a 1041 bp intervening sequence was present between the duplications; (3) Δ<it>recA </it>and Δ<it>recF </it>mutations resulted in lower frequencies of interplasmid recombination in Typhimurium and Paratyphi A, but not in Typhi; (4) in some cases, a Δ<it>recJ </it>mutation could reduce plasmid recombination but was less effective than Δ<it>recA </it>and Δ<it>recF </it>mutations. We also examined chromosome-related recombination. The frequencies of intrachromosomal recombination and plasmid integration into the chromosome were 2 and 3 logs lower than plasmid recombination frequencies in Rec⁺strains. A Δ<it>recA </it>mutation reduced both intrachromosomal recombination and plasmid integration frequencies.</p> <p>Conclusions</p> <p>The Δ<it>recA </it>and Δ<it>recF </it>mutations can reduce plasmid recombination frequencies in <it>Salmonella enterica</it>, but the effect can vary between serovars. This information will be useful for developing <it>Salmonella </it>delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.</p

Pre-pandemic SARS-CoV-2-specific IFN-γ and antibody responses were low in Ugandan samples and significantly reduced in HIV-positive specimens

IntroductionWe investigated whether prior SARS-CoV-2-specific IFN-γ and antibody responses in Ugandan COVID-19 pre-pandemic specimens aligned to this population's low disease severity.MethodsWe used nucleoprotein (N), spike (S), NTD, RBD, envelope, membrane, SD1/2-directed IFN-γ ELISpots, and an S- and N-IgG antibody ELISA to screen for SARS-CoV-2-specific cross-reactivity.ResultsHCoV-OC43-, HCoV-229E-, and SARS-CoV-2-specific IFN-γ occurred in 23, 15, and 17 of 104 specimens, respectively. Cross-reactive IgG was more common against the nucleoprotein (7/110, 15.5%; p = 0.0016, Fishers' Exact) than the spike (3/110, 2.72%). Specimens lacking anti-HuCoV antibodies had higher rates of pre-epidemic SARS-CoV-2-specific IFN-γ cross-reactivity (p-value = 0.00001, Fishers’ exact test), suggesting that exposure to additional factors not examined here might play a role. SARS-CoV-2-specific cross-reactive antibodies were significantly less common in HIV-positive specimens (p=0.017; Fishers' Exact test). Correlations between SARS-CoV-2- and HuCoV-specific IFN-γ responses were consistently weak in both HIV negative and positive specimens.DiscussionThese findings support the existence of pre-epidemic SARS-CoV-2-specific cellular and humoral cross-reactivity in this population. The data do not establish that these virus-specific IFN-γ and antibody responses are entirely specific to SARS-CoV-2. Inability of the antibodies to neutralise SARS-CoV-2 implies that prior exposure did not result in immunity. Correlations between SARS-CoV-2 and HuCoV-specific responses were consistently weak, suggesting that additional variables likely contributed to the pre-epidemic cross-reactivity patterns. The data suggests that surveillance efforts based on the nucleoprotein might overestimate the exposure to SARS-CoV-2 compared to inclusion of additional targets, like the spike protein. This study, while limited in scope, suggests that HIV-positive people are less likely than HIV-negative people to produce protective antibodies against SARS-CoV-2

The number of bacteria positive for carbapenemase genes and number of genes per organism.

<p>The number of bacteria positive for carbapenemase genes and number of genes per organism.</p

Susceptibilitypattern of bacteria used in the study.

<p>AMC-Amoxycillin clavulanate</p><p>SXT-Trimethoprimsulfamethoxazole</p><p>CXM-Cefuroxime</p><p>TEM-Temocillin</p><p>TPZ-tazobactumpipperacillin</p><p>FOX-Cefoxitin</p><p>FEP-Cefipime</p><p>CRO-Ceftriaxone</p><p>CAZ-Ceftazidime</p><p>CTX-Cefotaxime</p><p>MEM-Meropenem.</p><p>Susceptibilitypattern of bacteria used in the study.</p