ABSTRACT
The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination-inhibition (HA/HI) tests are well-known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 is unlikely to agglutinate to human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated enzyme-linked immunosorbent assay (ELISA) or cell-based assays such as the microneutralization assay. In this study, we developed the particle agglutination/particle agglutination-inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The PA titer was positively correlated with the plaque-forming units. The PAI titer using post-infection Syrian hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The results show the PAI test is useful for easy and rapid antigenic analysis of SARS-CoV-2.
ABSTRACT
The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.
ABSTRACT
To control the coronavirus disease 2019 (COVID-19) pandemic, there is a need to develop vaccines to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. One candidate is a nasal vaccine capable of inducing secretory IgA antibodies in the mucosa of the upper respiratory tract, the initial site of infection. However, regarding the development of COVID-19 vaccines, there is concern about the potential risk of inducing lung eosinophilic immunopathology as a vaccine-associated enhanced respiratory disease as a result of the T helper 2 (Th2)-dominant adaptive immune response. In this study, we investigated the protective effect against virus infection induced by intranasal vaccination of recombinant trimeric spike protein derived from SARS-CoV-2 adjuvanted with CpG oligonucleotides, ODN2006, in mouse model. The intranasal vaccine combined with ODN2006 successfully induced not only systemic spike-specific IgG antibodies, but also secretory IgA antibodies in the nasal mucosa. Secretory IgA antibodies showed high protective ability against SARS-CoV-2 variants (Alpha, Beta and Gamma variants) compared to IgG antibodies in the serum. The nasal vaccine of this formulation induced a high number of IFN-{gamma}-secreting cells in the draining cervical lymph nodes and a lower spike-specific IgG1/IgG2a ratio compared to that of subcutaneous vaccination with alum as a typical Th2 adjuvant. These features are consistent with the induction of the Th1 adaptive immune response. In addition, mice intranasally vaccinated with ODN2006 showed less lung eosinophilic immunopathology after viral challenge than mice subcutaneously vaccinated with alum adjuvant. Our findings indicate that intranasal vaccine adjuvanted with ODN2006 could be a candidate that can prevent the infection of antigenically different variant viruses, reducing the risk of vaccine-associated enhanced respiratory disease.
ABSTRACT
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergences impact on choosing optimal drug treatment.
ABSTRACT
BackgroundThe immune profile against SARS-CoV-2 has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by the Omicron in individuals with various immune histories. MethodsThe neutralization susceptibility of the variants including the Omicron and their ancestor was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections by the Alpha/Delta with multiple time intervals following vaccination. FindingsThe Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against the Omicron were induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. ConclusionsImmune histories with breakthrough infections can overcome the resistance to infection by the Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against the Omicron and future variants. FundingThis study was supported by grants from the Japan Agency for Medical Research and Development (AMED).
ABSTRACT
Prominent genomic recombination has been observed between the Delta and Alpha variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from clinical specimens in Japan. It is necessary to intensively study such marked genetic variations and characterize the emerging variants after careful verification of their lineage and clade assignment.
ABSTRACT
Rapid diagnosis of COVID-19 is essential for instituting measures to prevent viral spread. SARS-CoV-2 antigen rapid diagnostic test (Ag-RDT) based on lateral flow immunochromatography assay (LFIA) principle can visually indicate the presence of SARS-CoV-2 antigens as a band. Ag-RDT is clinically promising as a point-of-care testing because it can give results in a short time without the need for special equipment. Although various antigen capture LFIAs are now available for rapid diagnosis for SARS-CoV-2 infection, they face the problems of low sensitivity. We have previously developed highly specific monoclonal antibodies (mAb) against SARS-CoV-2 nucleocapsid protein (NP) and in this study, we have employed these mAbs to develop a new LFIA that can detect SARS-CoV-2 NP in nasopharyngeal swab samples with higher sensitivity by combining them with silver amplification technology. We also compared the performance of our Ag-RDT against the commercially available Ag-RDTs using clinical samples to find that our newly developed LFIA performed best among tested, highlighting the superiority of silver amplification technology.
ABSTRACT
Coronavirus disease 2019 (COVID-19) has had a major disease burden on many countries around the world. The spread of COVID-19 is anticipated to have a major impact on developing countries including African nations. To establish a point-of-care test for COVID-19, we developed a dry loop-mediated isothermal amplification (LAMP) method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. We carried out reverse transcription (RT)-LAMP using the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan). The entire mixture except for the primers is dried and immobilized inside the tube lid. To determine the specificity of the kit, 22 viral genomes associated with respiratory infections, including the SARS coronavirus, were tested. No LAMP product was detected in reactions performed with RNA from these pathogens. The sensitivity of this assay, determined by either a real-time turbidity assay or colorimetric change of the reaction mixture, as evaluated by the naked eye or under illumination with ultraviolet light, was 10 copies/reaction. After the initial validation analysis, we analyzed 24 nasopharyngeal swab specimens collected from patients suspected to have COVID-19. Nineteen (79.2%) of the 24 samples were positive for SARS-CoV-2 RNA, as determined by real-time RT-PCR analysis. Using the Loopamp SARS-CoV-2 Detection kit, we detected SARS-CoV-2 RNA in 15 (62.5%) of the 24 samples. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp 2019-CoV-2 detection reagent kit were 94.0%, 96.0%, 95.9%, and 94.1%, respectively. The dry LAMP method for detection of SARS-CoV-2 RNA was fast and easy to use, solves the cold chain problem, and therefore represents a promising tool for diagnosis of COVID-19 in developing countries. Author summaryCoronavirus disease 2019 (COVID-19) is a major public health problem around the world. A reliable point-of-care (POC) test for severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is urgently needed, especially in developing countries. The loop-mediated isothermal amplification (LAMP) method amplifies template nucleotides under isothermal conditions with high efficiency and specificity, both of which are major advantages for a POC test. In addition, because dry LAMP reagents can be stored at 4{degrees}C, it is suitable for use in developing countries. We evaluated the specificity and sensitivity of the Loopamp SARS-CoV-2 Detection kit (Eiken Chemical, Tokyo, Japan), a dry LAMP method for amplifying viral RNA. The initial validation study revealed that the method was highly specific and sensitive (lower detection limit: 10 copies/reaction). We then analyzed 24 nasopharyngeal swab specimens from patients suspected to have COVID-19. Using the Loopamp SARS-CoV-2 Detection kit, SARS-CoV-2 RNA was detected in 15 (62.5%) of the 24 samples. Compared with the standard real-time reverse transcription PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the Loopamp SARS-CoV-2 Detection kit were 78.9%, 100%, 100%, and 55.6%, respectively.
ABSTRACT
SARS-CoV-2 neutralizing antibodies confer protective immunity against reinfection. We have developed a rapid test for screening SARS-CoV-2 neutralization antibodies using genome-free virus-like particles incorporated with a small luciferase peptide, HiBiT. Their entry into LgBiT-expressing target cells reconstitutes NanoLuc luciferase readily detected by a luminometer. This newly developed HiBiT-tagged Virus-like particle-based Neutralization Test (hiVNT) can readily quantify SARS-CoV-2 neutralizing antibodies within three hours with a high-throughput in a low biosafety setting. Moreover, the neutralizing activity obtained from hiVNT was highly consistent with that measured by the conventional neutralization test using authentic SARS-CoV-2. Furthermore, antibody responses to both viral spike and nucleocapsid proteins correlated with the neutralization activity assessed by hiVNT. Our newly-developed hiVNT could be instrumental to survey individuals for the presence of functional neutralizing antibody against SARS-CoV-2.
ABSTRACT
The Diamond Princess (DP) cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a COVID-19 case. We performed whole genome sequencing of SARS-CoV-2 directly from PCR-positive clinical specimens and conducted a haplotype network analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the DP originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters were linked to transmission through mass-gathering events in the recreational areas and direct transmission among passengers who shared cabins during the quarantine. This study demonstrates the usefulness of haplotype network analysis in identifying potential infection routes. One Sentence SummaryGenome-based tracing of SARS-CoV-2 infections among passengers and crews in Diamond Princess cruise ship during the quarantine
