Fatal COVID-19 outcomes are associated with an antibody response targeting epitopes shared with endemic coronaviruses.

The role of immune responses to previously seen endemic coronavirus epitopes in severe acute respiratory coronavirus 2 (SARS-CoV-2) infection and disease progression has not yet been determined. Here, we show that a key characteristic of fatal outcomes with coronavirus disease 2019 (COVID-19) is that the immune response to the SARS-CoV-2 spike protein is enriched for antibodies directed against epitopes shared with endemic beta-coronaviruses and has a lower proportion of antibodies targeting the more protective variable regions of the spike. The magnitude of antibody responses to the SARS-CoV-2 full-length spike protein, its domains and subunits, and the SARS-CoV-2 nucleocapsid also correlated strongly with responses to the endemic beta-coronavirus spike proteins in individuals admitted to an intensive care unit (ICU) with fatal COVID-19 outcomes, but not in individuals with nonfatal outcomes. This correlation was found to be due to the antibody response directed at the S2 subunit of the SARS-CoV-2 spike protein, which has the highest degree of conservation between the beta-coronavirus spike proteins. Intriguingly, antibody responses to the less cross-reactive SARS-CoV-2 nucleocapsid were not significantly different in individuals who were admitted to an ICU with fatal and nonfatal outcomes, suggesting an antibody profile in individuals with fatal outcomes consistent with an "original antigenic sin" type response.

Development of Viral-Vectored Vaccines and Virus Replicon Particle-Based Neutralisation Assay against Mayaro Virus.

Mayaro virus (MAYV) is an emerging alphavirus causing acute febrile illness associated with chronic polyarthralgia. Although MAYV is currently restricted to tropical regions in South America around the Amazon basin, it has the potential to spread globally by Aedes species mosquitoes. In addition, there are currently no specific therapeutics or licenced vaccines against MAYV infection. We have previously shown that an adenovirus based Mayaro vaccine (ChAdOx1 May) was able to provide full protection against MAYV challenge in vaccinated A129 mice and induced high neutralising antibody titres. In this study, we have constructed a replication deficient simian adenovirus (ChAdOx2) and a Modified Ankara Virus (MVA) based vaccine expressing the MAYV structural cassette (sMAYV) similar to ChAdOx1 May, and characterised recombinant MAYV E2 glycoprotein expressed in a mammalian system for immune monitoring. We demonstrate that ChAdOx2 May was able to induce high antibody titres similar to ChAdOx1 May, and MVA May was shown to be an effective boosting strategy following prime vaccination with ChAdOx1 or ChAdOx2 May. In order to measure MAYV neutralising ability, we have developed a virus replicon particle-based neutralisation assay which effectively detected neutralising antibodies against MAYV. In summary, our study indicates the potential for further clinical development of the viral vectored MAYV vaccines against MAYV infections.

A universal vaccine candidate against Plasmodium vivax malaria confers protective immunity against the three PvCSP alleles.

Malaria is a highly prevalent parasitic disease in regions with tropical and subtropical climates worldwide. Among the species of Plasmodium causing human malaria, P. vivax is the second most prevalent and the most geographically widespread species. A major target of a pre-erythrocytic vaccine is the P. vivax circumsporozoite protein (PvCSP). In previous studies, we fused two recombinant proteins representing three allelic variants of PvCSP (VK210, VK247 and P. vivax-like) to the mumps virus nucleocapsid protein to enhance immune responses against PvCSP. The objective of the present study was to evaluate the protective efficacy of these recombinants in mice challenged with transgenic P. berghei parasites expressing PvCSP allelic variants. Formulations containing Poly (I:C) or Montanide ISA720 as adjuvants elicited high and long-lasting IgG antibody titers specific to each PvCSP allelic variant. Immunized mice were challenged with two existing chimeric P. berghei parasite lines expressing PvCSP-VK210 and PvCSP-VK247. We also developed a novel chimeric line expressing the third allelic variant, PvCSP-P. vivax-like, as a new murine immunization-challenge model. Our formulations conferred partial protection (significant delay in the time to reach 1% parasitemia) against challenge with the three chimeric parasites. Our results provide insights into the development of a vaccine targeting multiple strains of P. vivax.

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone progressive change, with variants conferring advantage rapidly becoming dominant lineages, e.g., B.1.617. With apparent increased transmissibility, variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the United Kingdom. Here we study the ability of monoclonal antibodies and convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2, complement this with structural analyses of Fab/receptor binding domain (RBD) complexes, and map the antigenic space of current variants. Neutralization of both viruses is reduced compared with ancestral Wuhan-related strains, but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2, suggesting that individuals infected previously by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insights for immunization policy with future variant vaccines in non-immune populations.

Platelet activation and aggregation response to dengue virus nonstructural protein 1 and domains.

BACKGROUND: Platelets are now recognized as immunological sentries in the first line of defense that participate in the detection and response to pathogens. This frequently results in a decrease in the number of circulating platelets. Different mechanisms have been hypothesized to explain the thrombocytopenia in patients with severe dengue, one of them is the participation of the non-structural protein 1 (NS1) of dengue virus (DENV), which can be secreted into circulation during DENV infection and promotes a more efficient infection. OBJECTIVE: The present study aimed to investigate the ability of platelet response to stimulation with full-length DENV NS1 protein and its domains. METHODS: DENV NS1 plasmid was transfected into HEK-293T. Proteins were purified by Niquel Sepharose affinity chromatography. Secreted proteins were assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis, Coomassie staining and western blot. Platelet-rich plasma was directly incubated with DENV NS1 proteins. Platelet activation was confirmed by expression of αIIbßIII and P-selectin by flow cytometry. Platelet aggregation was also assessed using DENV NS1 protein and its individual domains as agonists. RESULTS: DENV NS1 protein and its domains induce P-selectin and αIIbß3 complex expression on platelet surfaces. DENV NS1 induce a stable platelet aggregation after the addition of a minimal dose of adenosine diphosphate (ADP), epinephrine (EPI), or collagen. Interestingly, only EPI could induce the formation of platelet aggregates after incubation with the protein domains of NS1. CONCLUSION: Our results suggest that the full DENV NS1 protein and also its domains promote platelet recognition, activation, and aggregation.

Antibody evasion by the P.1 strain of SARS-CoV-2.

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations, including P.1 from Brazil, B.1.351 from South Africa, and B.1.1.7 from the UK (12, 10, and 9 changes in the spike, respectively). All have mutations in the ACE2 binding site, with P.1 and B.1.351 having a virtually identical triplet (E484K, K417N/T, and N501Y), which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine-induced antibody responses than B.1.351, suggesting that changes outside the receptor-binding domain (RBD) impact neutralization. Monoclonal antibody (mAb) 222 neutralizes all three variants despite interacting with two of the ACE2-binding site mutations. We explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

The antigenic anatomy of SARS-CoV-2 receptor binding domain.

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.

Reduced neutralization of SARS-CoV-2 B.1.1.7 variant by convalescent and vaccine sera.

SARS-CoV-2 has caused over 2 million deaths in little over a year. Vaccines are being deployed at scale, aiming to generate responses against the virus spike. The scale of the pandemic and error-prone virus replication is leading to the appearance of mutant viruses and potentially escape from antibody responses. Variant B.1.1.7, now dominant in the UK, with increased transmission, harbors 9 amino acid changes in the spike, including N501Y in the ACE2 interacting surface. We examine the ability of B.1.1.7 to evade antibody responses elicited by natural SARS-CoV-2 infection or vaccination. We map the impact of N501Y by structure/function analysis of a large panel of well-characterized monoclonal antibodies. B.1.1.7 is harder to neutralize than parental virus, compromising neutralization by some members of a major class of public antibodies through light-chain contacts with residue 501. However, widespread escape from monoclonal antibodies or antibody responses generated by natural infection or vaccination was not observed.

Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and vaccine-induced sera.

The race to produce vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began when the first sequence was published, and this forms the basis for vaccines currently deployed globally. Independent lineages of SARS-CoV-2 have recently been reported: UK, B.1.1.7; South Africa, B.1.351; and Brazil, P.1. These variants have multiple changes in the immunodominant spike protein that facilitates viral cell entry via the angiotensin-converting enzyme-2 (ACE2) receptor. Mutations in the receptor recognition site on the spike are of great concern for their potential for immune escape. Here, we describe a structure-function analysis of B.1.351 using a large cohort of convalescent and vaccinee serum samples. The receptor-binding domain mutations provide tighter ACE2 binding and widespread escape from monoclonal antibody neutralization largely driven by E484K, although K417N and N501Y act together against some important antibody classes. In a number of cases, it would appear that convalescent and some vaccine serum offers limited protection against this variant.

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum (preprint)

A pesquisa aponta que o soro de pessoas previamente infectadas por outras cepas é menos potente contra esta variante viral. O problema é observado de forma marcante entre os indivíduos anteriormente infectados pela variante Gama, identificada originalmente em Manaus e atualmente dominante no Brasil, assim como pela variante Beta, detectada pela primeira vez na África do Sul. Nestes casos, a capacidade de neutralizar a cepa Delta é onze vezes menor. O soro de pessoas vacinadas também tem potência reduzida contra a variante originária da Índia, mas os dados apontam que as vacinas continuam efetivas. A capacidade de neutralizar a cepa é 2,5 vezes menor para o imunizante da Pfizer e 4,3 vezes menor para o da Astrazeneca. Os autores do trabalho ressaltam que os índices são semelhantes aos verificados com as variantes Gama e Alfa ­ que emergiram no Brasil e no Reino Unido, respectivamente. Não há evidência de fuga generalizada da neutralização, diferentemente do registrado com a variante Beta ­ com origem na África do Sul.

Adenoviral-Vectored Mayaro and Chikungunya Virus Vaccine Candidates Afford Partial Cross-Protection From Lethal Challenge in A129 Mouse Model.

Mayaro (MAYV) and chikungunya viruses (CHIKV) are vector-borne arthritogenic alphaviruses that cause acute febrile illnesses. CHIKV is widespread and has recently caused large urban outbreaks, whereas the distribution of MAYV is restricted to tropical areas in South America with small and sporadic outbreaks. Because MAYV and CHIKV are closely related and have high amino acid similarity, we investigated whether vaccination against one could provide cross-protection against the other. We vaccinated A129 mice (IFNAR -/-) with vaccines based on chimpanzee adenoviral vectors encoding the structural proteins of either MAYV or CHIKV. ChAdOx1 May is a novel vaccine against MAYV, whereas ChAdOx1 Chik is a vaccine against CHIKV already undergoing early phase I clinical trials. We demonstrate that ChAdOx1 May was able to afford full protection against MAYV challenge in mice, with most samples yielding neutralizing PRNT80 antibody titers of 1:258. ChAdOx1 May also provided partial cross-protection against CHIKV, with protection being assessed using the following parameters: survival, weight loss, foot swelling and viremia. Reciprocally, ChAdOx1 Chik vaccination reduced MAYV viral load, as well as morbidity and lethality caused by this virus, but did not protect against foot swelling. The cross-protection observed is likely to be, at least in part, secondary to cross-neutralizing antibodies induced by both vaccines. In summary, our findings suggest that ChAdOx1 Chik and ChAdOx1 May vaccines are not only efficacious against CHIKV and MAYV, respectively, but also afford partial heterologous cross-protection.

Detection of neutralising antibodies to SARS-CoV-2 to determine population exposure in Scottish blood donors between March and May 2020.

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon.

Circumsporozoite protein (CSP) variants of P. vivax, besides having variations in the protein repetitive portion, can differ from each other in aspects such as geographical distribution, intensity of transmission, vectorial competence and immune response. Such aspects must be considered to P. vivax vaccine development. Therefore, we evaluated the immunogenicity of novel recombinant proteins corresponding to each of the three P. vivax allelic variants (VK210, VK247 and P. vivax-like) and of the C-terminal region (shared by all PvCSP variants) in naturally malaria-exposed populations of Brazilian Amazon. Our results demonstrated that PvCSP-VK210 was the major target of humoral immune response in studied population, presenting higher frequency and magnitude of IgG response. The IgG subclass profile showed a prevalence of cytophilic antibodies (IgG1 and IgG3), that seem to have an essential role in protective immune response. Differently of PvCSP allelic variants, antibodies elicited against C-terminal region of protein did not correlate with epidemiological parameters, bringing additional evidence that humoral response against this protein region is not essential to protective immunity. Taken together, these findings increase the knowledge on serological response to distinct PvCSP allelic variants and may contribute to the development of a global and effective P. vivax vaccine.

Evaluation of Chimpanzee Adenovirus and MVA Expressing TRAP and CSP from Plasmodium cynomolgi to Prevent Malaria Relapse in Nonhuman Primates.

Plasmodium vivax is the world's most widely distributed human malaria parasite, with over 2.8 billion people at risk in Asia, the Americas, and Africa. The 80-90% new P. vivax malaria infections are due to relapses which suggest that a vaccine with high efficacy against relapses by prevention of hypnozoite formation could lead to a significant reduction in the prevalence of P. vivax infections. Here, we describe the development of new recombinant ChAdOx1 and MVA vectors expressing P. cynomolgi Thrombospondin Related Adhesive Protein (PcTRAP) and the circumsporozoite protein (PcCSP). Both were shown to be immunogenic in mice prior to their assessment in rhesus macaques. We confirmed good vaccine-induced humoral and cellular responses after prime-boost vaccination in rhesus macaques prior to sporozoite challenge. Results indicate that there were no significant differences between mock-control and vaccinated animals after challenge, in terms of protective efficacy measured as the time taken to 1st patency, or as number of relapses. This suggests that under the conditions tested, the vaccination with PcTRAP and PcCSP using ChAdOx1 or MVA vaccine platforms do not protect against pre-erythrocytic malaria or relapses despite good immunogenicity induced by the viral-vectored vaccines.

Broad and strong memory CD4 + and CD8 + T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFNγ based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4 + and/or CD8 + epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8 + T cells than spike-specific CD8 + T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8 + to CD4 + T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines.

The flavivirus envelope protein domain III (EDIII) was an effective immunogen against dengue virus (DENV) and other related flaviviruses. Whether this can be applied to the Zika virus (ZIKV) vaccinology remains an open question. Here, we tested the efficacy of ZIKV-EDIII against ZIKV infection, using several vaccine platforms that present the antigen in various ways. We provide data demonstrating that mice vaccinated with a ZIKV-EDIII as DNA or protein-based vaccines failed to raise fully neutralizing antibodies and did not control viremia, following a ZIKV challenge, despite eliciting robust antibody responses. Furthermore, we showed that ZIKV-EDIII encoded in replication-deficient Chimpanzee adenovirus (ChAdOx1-EDIII) elicited anti-ZIKV envelope antibodies in vaccinated mice but also provided limited protection against ZIKV in two physiologically different mouse challenge models. Taken together, our data indicate that contrary to what was shown for other flaviviruses like the dengue virus, which has close similarities with ZIKV-EDIII, this antigen might not be a suitable vaccine candidate for the correct induction of protective immune responses against ZIKV.

Production and Purification of Zika Virus NS1 Glycoprotein in HEK293 Cells.

In this chapter, we describe production and purification of the Zika virus NS1 glycoprotein in human embryonic kidney (HEK293T) cells at small, research laboratory scale. The expression of secreted NS1 (sNS1) and the C-terminal ß-ladder domain in HEK293T cells were tested in a small-scale transfection before scaling up to a larger-scale transfection using roller bottles. Two different purification approaches have been applied to obtain purified NS1 (sNS1) and the C-terminal ß-ladder domain ready for clinical applications.

Drug-cured experimental Trypanosoma cruzi infections confer long-lasting and cross-strain protection.

BACKGROUND: The long term and complex nature of Chagas disease in humans has restricted studies on vaccine feasibility. Animal models also have limitations due to technical difficulties in monitoring the extremely low parasite burden that is characteristic of chronic stage infections. Advances in imaging technology offer alternative approaches that circumvent these problems. Here, we describe the use of highly sensitive whole body in vivo imaging to assess the efficacy of recombinant viral vector vaccines and benznidazole-cured infections to protect mice from challenge with Trypanosoma cruzi. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with T. cruzi strains modified to express a red-shifted luciferase reporter. Using bioluminescence imaging, we assessed the degree of immunity to re-infection conferred after benznidazole-cure. Those infected for 14 days or more, prior to the onset of benznidazole treatment, were highly protected from challenge with both homologous and heterologous strains. There was a >99% reduction in parasite burden, with parasites frequently undetectable after homologous challenge. This level of protection was considerably greater than that achieved with recombinant vaccines. It was also independent of the route of infection or size of the challenge inoculum, and was long-lasting, with no significant diminution in immunity after almost a year. When the primary infection was benznidazole-treated after 4 days (before completion of the first cycle of intracellular infection), the degree of protection was much reduced, an outcome associated with a minimal T. cruzi-specific IFN-γ+ T cell response. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that a protective Chagas disease vaccine must have the ability to eliminate parasites before they reach organs/tissues, such as the GI tract, where once established, they become largely refractory to the induced immune response.

Antibody testing for COVID-19: A report from the National COVID Scientific Advisory Panel.

Background: The COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. Methods: We tested plasma for COVID (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). Results: ELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested ≥10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. Conclusions: Currently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.