A next-generation intranasal trivalent MMS vaccine induces durable and broad protection against SARS-CoV-2 variants of concern.

As SARS-CoV-2 variants of concern (VoCs) that evade immunity continue to emerge, next-generation adaptable COVID-19 vaccines which protect the respiratory tract and provide broader, more effective, and durable protection are urgently needed. Here, we have developed one such approach, a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. This vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2] that are known to provide safe, effective, and long-lasting protective immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and two important SARS-CoV-2 VoCs (Delta and Omicron BA.1) were each inserted into one of these three viruses which were then combined into a trivalent "MMS" candidate vaccine. Intranasal immunization of MMS in IFNAR1-/- mice induced a strong SARS-CoV-2-specific serum IgG response, cross-variant neutralizing antibodies, mucosal IgA, and systemic and tissue-resident T cells. Immunization of golden Syrian hamsters with MMS vaccine induced similarly high levels of antibodies that efficiently neutralized SARS-CoV-2 VoCs and provided broad and complete protection against challenge with any of these VoCs. This MMS vaccine is an efficacious, broadly protective next-generation COVID-19 vaccine candidate, which is readily adaptable to new variants, built on a platform with a 50-y safety record that also protects against measles and mumps.

Recombinant measles virus expressing prefusion spike protein stabilized by six rather than two prolines is more efficacious against SARS-CoV-2 infection.

Measles virus (MeV) has been an excellent vector platform for delivering vaccines against many pathogens because of its high safety and efficacy, and induction of long-lived immunity. Early in the COVID-19 pandemic, a recombinant MeV (rMeV) expressing the prefusion full-length spike protein stabilized by two prolines (TMV-083) was developed and tested in phase 1 and 1/2 clinical trials but was discontinued because of insufficient immunogenicity and a low seroconversion rate in adults. Here, we compared the immunogenicity of rMeV expressing a soluble prefusion spike (preS) protein stabilized by two prolines (rMeV-preS-2P) with a rMeV expressing a soluble preS protein stabilized by six prolines (rMeV-preS-6P). We found that rMeV-preS-6P expressed approximately five times more preS than rMeV-preS-2P in cell culture. Importantly, rMeV-preS-6P induced 30-60 and six times more serum immunoglobulin G and neutralizing antibody than rMeV-preS-2P, respectively, in IFNAR-/- mice. IFNAR-/- mice immunized with rMeV-preS-6P were completely protected from challenge with a mouse-adapted SARS-CoV-2, whereas those immunized with rMeV-preS-2P were partially protected. In addition, hamsters immunized with rMeV-preS-6P were completely protected from the challenge with a Delta variant of SARS-CoV-2. Our results demonstrate that rMeV-preS-6P is significantly more efficacious than rMeV-preS-2P, highlighting the value of using preS-6P as the antigen for developing vaccines against SARS-CoV-2.

Three SARS-CoV-2 spike protein variants delivered intranasally by measles and mumps vaccines are broadly protective.

As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1-/- mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.

Epidemiology and clinical characteristics of severe acute respiratory infections among hospitalized children under 5 years of age in a tertiary care center in Bangkok, Thailand, 2019-2020.

Background: Acute respiratory infections (ARIs) are common in children and can range in severity from mild self-limiting illnesses to more severe conditions such as pneumonia and respiratory failure. Data on the epidemiology of viral and bacterial pathogens causing ARIs in children are scarce in this region. This study aimed to investigate the epidemiology and clinical manifestations of pathogens in children aged ≤5 years presenting with severe acute respiratory infection (SARI) in Bangkok, Thailand. The impact of rapid multiplex PCR-based testing on clinical management is also explored. Methods: This cross-sectional study enrolled consecutive children aged ≤5 years presenting with SARI at a tertiary care centre in Bangkok, Thailand, between 2019 and 2020. Nasopharyngeal swabs were collected once at admission, and viral and bacterial pathogens were tested using the QIAstat-Dx respiratory panel. Results: A total of 169 children were enrolled in this study. At least one pathogenic virus was detected in 91.7 % of participants. Based on the final diagnoses made upon discharge, 30.2 % had upper respiratory tract infection, whereas 66.3 % had lower respiratory tract infection. Pneumonia was the most common diagnosis (59.2 %). The most common pathogen identified was rhino/enterovirus (45.2 %), followed by respiratory syncytial virus (31.6 %) and parainfluenza virus (14.2 %). Co-infection was found in 15.4 % and was not associated with increased disease severity. Conclusions: This study provides additional insights into the pathogen profiles, clinical diagnosis, and co-infection combinations of ARIs in hospitalized children. This information is useful for diagnosis and treatment of ARIs, as well as implementation of appropriate infection control measures and guidance for future vaccine policy development.

High seroprevalence of antibodies against human respiratory syncytial virus and evidence of respiratory syncytial virus reinfection in young children in Thailand.

OBJECTIVES: To investigate the seroprevalence of respiratory syncytial virus (RSV) infections in young children, the correlation between RSV antibody levels in maternal and cord serum, and to provide evidence of RSV reinfection in Thai children after primary infections. METHODS: Serum samples were collected from 302 mothers and 291 children between 2015 and 2021. Maternal and cord blood were collected at birth. Serial serum samples of children were collected at the ages of 2, 7, 18, 19, 24, 36, 48, and 60 months and the presence of anti-RSV immunoglobulin G (IgG) was tested using an enzyme-linked immunosorbent assay. RESULTS: The cord: maternal serum antibody ratio was 1.09 (95% confidence interval 1.08-1.11). Although >90% of babies at birth were seropositive through transplacental transfer, antibody levels gradually declined, with the highest seronegative rate (91.9%) at 7 months of age. Subsequently, anti-RSV IgG levels increased with age, most likely due to natural infection. One-third of the children showed evidence of reinfection as determined by seroconversion of anti-RSV IgG or increased titers of at least 50 relative units/ml. CONCLUSION: Waning of RSV antibodies in infants is rapid, and RSV infection subsequently increases anti-RSV IgG titers. RSV vaccination in children before the age of 7 months should be recommended.

Prescreening with a Rapid Diagnostic Test Followed by a Confirmatory Qualitative Nucleic Acid Test Can Simplify Hepatitis C Diagnosis.

Asymptomatic hepatitis C virus (HCV) infection without treatment is associated with chronic liver diseases including hepatocellular carcinoma. A major obstacle to hepatitis C diagnosis leading to antiviral treatment in some developing countries is the complicated HCV testing required before treatment. To simplify an HCV test-to-treat strategy, which could lead to timely diagnosis and treatment at the point-of-care, we evaluated the performance of four anti-HCV rapid diagnostic tests (RDTs) (Abon, Blue Cross, Healgen, and SD Bioline). They yielded comparable sensitivity (80-83%), specificity (99-100%), and accuracy (90-91.5%). When we field-tested Abon in 4,769 residents of an HCV-endemic province in Thailand, 306 seropositive individuals (6.4%) were identified. In comparison, laboratory test using an automated commercial chemiluminescent microparticle immunoassay (Abbott ARCHITECT) identified slightly more seropositives (327% or 6.9%). Field implementation suggests that Abon was sensitive (88.7%), specific (99.6%), and accurate (98.9%). Furthermore, 82% (250/306) of Abon-positive samples had detectable HCV RNA as determined by nucleic acid test (Roche cobas). The same 250 samples out of 327 reactive in Abbott immunoassay also had detectable HCV RNA (mean RNA level: log 6.28 IU/mL, range: log 3.06- 7.78 IU/mL). The use of RDT followed by qualitative nucleic acid test can cost-effectively identify the majority of HCV seropositive individuals with active infection, which will obviate the need for expensive viral load quantification tests when simplifying HCV diagnosis for the test-to-treat program at the point-of-care.

The impact of COVID-19 and control measures on public health in Thailand, 2020.

BACKGROUND: The COVID-19 virus has been an emerging disease causing global outbreaks for over a year. In Thailand, transmission may be controlled by strict measures that could positively and negatively impact physical health and suicidal behavior. METHODS: The incidence of COVID-19 was retrieved from the Department of Disease Control (DDC). The impact of viral diseases was retrieved from the open-source of the DDC and King Chulalongkorn Memorial Hospital. The road accidents data were from the Thai Ministry of Transport. The suicidal behavior data were obtained from the Department of Mental Health. We compared data from the year 2019 with the pandemic COVID-19 outbreak period in 2020, before lockdown, during lockdown, easing, and new wave period using unpaired t-test and least-squares linear regression. We compared the impact of the outbreak on various data records in 2020 with corresponding non-outbreak from 2019. RESULTS: There was a significant decline in cases of influenza (p < 0.001) and norovirus (p = 0.01). However, there was no significant difference in RSV cases (p = 0.17). There was a dramatic increase in attempt to suicides and suicides (p < 0.001). There was no impact on roadside accidents and outpatient department visits. DISCUSSION: The extensive intervention measures during lockdown during the first wave positively impacted total cases for each period for acute respiratory and gastrointestinal tract diseases, car accidents, and injuries and negatively impacted indicators of suicidal behavior. The data support government policies that would be effective against the next outbreak by promoting the "new normal" lifestyle.

Upsurge of human rhinovirus infection followed by a delayed seasonal respiratory syncytial virus infection in Thai children during the coronavirus pandemic.

BACKGROUND: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) commonly cause influenza-like illness in young children. The global coronavirus pandemic beginning in 2020 altered the seasonality and prevalence of these respiratory infections in Thailand. We aimed to characterize the upsurge of HRV and the subsequent RSV infection observed among young children who sought medical care at a hospital in Bangkok. METHODS: From July to December 2020, nasopharyngeal swabs from children ≤5 years of age presented with influenza-like illness were tested for RSV and HRV using reverse-transcription polymerase chain reaction. Positive samples were Sanger sequenced. Genotyping was performed using sequence and phylogenetic analysis. RESULTS: Upsurge of HRV infection began in July and was subsequently replaced by a surge of RSV infection from September onward. In 6 months, HRV was detected in 27.5% (158/574) of the samples, of which 44% (69/158) were HRV-A, 7% (11/158) were HRV-B, and 36% (57/158) were HRV-C. Meanwhile, RSV was detected in 40.4% (232/574) of the samples, of which 78% (181/232) were RSV-A and 6% (14/232) were RSV-B. RSV peaked in October 2020, approximately 2 months later than typically seen in previous years. All RSV-A were of subgenotype ON1. Codetection of HRV and RSV was found in 5.1% (29/574). CONCLUSIONS: HRV and RSV infection among young children coincided with relaxed local coronavirus public health measures, including the return to in-class schooling. The delayed RSV season in 2020 was predominantly associated with RSV-A.

Respiratory syncytial virus infection trend is associated with meteorological factors.

Respiratory syncytial virus (RSV) infects young children and causes influenza-like illness. RSV circulation and prevalence differ among countries and climates. To better understand whether climate factors influence the seasonality of RSV in Thailand, we examined RSV data from children ≤ 5 years-old who presented with respiratory symptoms from January 2012-December 2018. From a total of 8,209 nasopharyngeal samples, 13.2% (1,082/8,209) was RSV-positive, of which 37.5% (406/1,082) were RSV-A and 36.4% (394/1,082) were RSV-B. The annual unimodal RSV activity from July-November overlaps with the rainy season. Association between meteorological data including monthly average temperature, relative humidity, rainfall, and wind speed for central Thailand and the incidence of RSV over 7-years was analyzed using Spearman's rank and partial correlation. Multivariate time-series analysis with an autoregressive integrated moving average (ARIMA) model showed that RSV activity correlated positively with rainfall (r = 0.41) and relative humidity (r = 0.25), but negatively with mean temperature (r = - 0.27). The best-fitting ARIMA (1,0,0)(2,1,0)12 model suggests that peak RSV activity lags the hottest month of the year by 4 months. Our results enable possible prediction of RSV activity based on the climate and could help to anticipate the yearly upsurge of RSV in this region.

Prevalence of Hepatitis C Virus in an Endemic Area of Thailand: Burden Assessment toward HCV Elimination.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. To eliminate HCV infection in an endemic area, an epidemiological baseline of the current HCV infection in the population is required. We therefore aimed to evaluate the HCV burden in the Thai Province of Phetchabun, which has the highest HCV infection rate in the country. Toward this, a province-wide district-based representative sampling of 4,769 individuals ages 35-64 years previously shown to represent high-risk age-groups were tested for anti-HCV antibodies using the automated chemiluminescent microparticle assays. Active HCV infection and subsequent genotyping were determined from serologically reactive samples by amplification of the HCV core gene. We found that 6.9% (327/4,769) were anti-HCV positive, of which 75.8% (248/327) had detectable HCV RNA and 5.8% (19/327) were in the presence of hepatitis B virus coinfection. Nucleotide sequencing and phylogenetic analysis revealed that HCV genotype 6 was the most prevalent (41%, 101/248), followed by genotype 3 (31%, 78/248), and genotype 1 (28%, 69/248). Socioeconomic and demographic factors including male gender, education, and agricultural work were associated with HCV seropositivity. From these results, we defined the regional HCV genotypes and estimated the HCV burden necessary toward the implementation of pan-genotypic direct-acting antivirals, which may be appropriate and effective toward the diversity of genotypes identified in this study. Micro-elimination of HCV in Phetchabun may serve as a model for a more comprehensive coverage of HCV treatment in Thailand.

Respiratory syncytial virus, human metapneumovirus, and influenza virus infection in Bangkok, 2016-2017.

Children and adults residing in densely populated urban centers around the world are at risk of seasonal influenza-like illness caused by respiratory viruses such as influenza virus, human metapneumovirus (hMPV), and respiratory syncytial virus (RSV). In a large metropolitan of Thailand's capital city Bangkok, most respiratory infections are rarely confirmed by molecular diagnostics. We therefore examined the frequency of RSV, hMPV, and influenza virus in 8,842 patients who presented influenza-like illness and sought medical care at a large hospital in Bangkok between 2016 and 2017. Using a multiplex real-time reverse-transcription polymerase chain reaction (RT-PCR), 30.5% (2,699/8,842) of nasopharyngeal (NP) swab samples tested positive for one or more of these viruses. Influenza virus comprised 17.3% (1,528/8,842), of which the majority were influenza A/H3N2. Such infection was most prevalent among adults and the elderly. RSV was identified in 11.4% (1,011/8,842) and were mostly ON1 and BA9 genotypes. Of the hMPV-positive samples (3.6%, 318/8,842), genotypes A2, B1, and B2 were detected. A small number of individuals experienced co-infections (1.8%, 155/8,842), most commonly between RSV and influenza A/H3N2. RSV and hMPV co-infections were also found, but mainly in young children. Viral respiratory tract infection peaked locally in the rainy season (June to September). These findings support the utility of rapid nucleic acid testing of RSV, hMPV, and influenza virus in patients with ILI.

Respiratory syncytial virus genotypes NA1, ON1, and BA9 are prevalent in Thailand, 2012-2015.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infection in infants and young children worldwide. To investigate the RSV burden in Thailand over four consecutive years (January 2012 to December 2015), we screened 3,306 samples obtained from children ≤5 years old with acute respiratory tract infection using semi-nested reverse-transcription polymerase chain reaction (RT-PCR). In all, 8.4% (277/3,306) of the specimens tested positive for RSV, most of which appeared in the rainy months of July to November. We then genotyped RSV by sequencing the G glycoprotein gene and performed phylogenetic analysis to determine the RSV antigenic subgroup. The majority (57.4%, 159/277) of the RSV belonged to subgroup A (RSV-A), of which NA1 genotype was the most common in 2012 while ON1 genotype became prevalent the following year. Among samples tested positive for RSV-B subgroup B (RSV-B) (42.6%, 118/277), most were genotype BA9 (92.6%, 87/94) with some BA10 and BA-C. Predicted amino acid sequence from the partial G region showed highly conserved N-linked glycosylation site at residue N237 among all RSV-A ON1 strains (68/68), and at residues N296 (86/87) and N310 (87/87) among RSV-B BA9 strains. Positive selection of key residues combined with notable sequence variations on the G gene contributed to the continued circulation of this rapidly evolving virus.

Prevalence and Phylogenetic Characterization of Enterovirus D68 in Pediatric Patients with Acute Respiratory Tract Infection in Thailand.

Enterovirus D68 (EV-D68) is associated with severe lower respiratory tract infection and neurological abnormalities including acute myelitis and cranial nerve dysfunction. To determine whether an increased incidence of EV-D68 occurs in Southeast Asia, we retrospectively tested specimens collected from Thai pediatric patients who were less than 5 years of age and presented with acute respiratory tract infections between 2012 and 2014. Reverse transcription-polymerase chain reaction and nucleotide sequencing of the 5'-UTR/VP2 region were used to identify EV-D68. We also examined the epidemiological pattern of EV-D68 since 2009, when it was first identified in Thailand, and compiled records of clinical manifestations in children with confirmed EV-D68 infection. From 837 samples, 5 samples (0.6%) tested positive for EV-D68. All patients presented with viral pneumonia and required hospitalization. Phylogenetic analysis of the VP4/VP2 regions revealed that EV-D68 strains circulating in Thailand between 2012 and 2014 were closely related to strains reported in Japan, United Kingdom, China, and France. Continued surveillance of probable EV-D68-associated severe respiratory tract infection and the development of a rapid diagnostic test for EV-D68 are essential in supporting awareness and facilitating disease prevention and control.

Molecular epidemiology and characterization of human coronavirus in Thailand, 2012-2013.

BACKGROUND: Coronavirus causes respiratory infections in humans. To determine the prevalence of human coronavirus (HCoV) infection among patients with influenza-like illness, 5833 clinical samples from nasopharyngeal swabs and aspirates collected between January 2012 and December 2013 were examined. RESULTS: HCoV was found in 46 (0.79 %) samples. There were 19 (0.32 %) HCoV-HKU1, 19 (0.32 %) HCoV-NL63, 5 (0.09 %) HCoV-229E, and 3 (0.05 %) HCoV-OC43. None of the sample tested positive for MERS-CoV. The majority (54 %) of the HCoV-positive patients were between the ages of 0 and 5 years. HCoV was detected throughout the 2-year period and generally peaked from May to October, which coincided with the rainy season. Phylogenetic trees based on the alignment of the spike (S) gene sequences suggest an emergence of a new clade for HCoV-229E. CONCLUSIONS: The data in this study provide an insight into the prevalence of the recent circulating HCoVs in the region.