Intranasal immunization with the bivalent SARS-CoV-2 vaccine effectively protects mice from nasal infection and completely inhibits disease development.

With the continuous emergence of coronavirus disease 2019 (COVID-19) waves, the scientific community has developed a vaccine that offers broad-spectrum protection at virus-targeted organs for inhibiting the transmission and protection of disease development. In the present study, a bivalent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine containing receptor-binding domain (RBD) protein of spike from Wuhan-1 and omicron BA.1 loaded in nanoparticles, bivalent RBD NPs, was developed. The immunogenicity and protective efficacy of this vaccine candidate were evaluated using an in vivo model. Results showed that mice that received intranasal cGAMP-adjuvanted bivalent RBD-NPs vaccine elicited robust and durable antibody responses. The stimulated antibody broadly neutralized the ancestral strain and variants of concerns (delta and omicron BA.1) in the upper and lower respiratory tracts. Furthermore, the immunized mice developed T-cell response in their lung tissue. Importantly, intranasal immunization with this vaccine candidate efficiently protected mice from nasal infection caused by both Wuhan-1 and BA.1 viruses. Immunized mice that remained susceptible to nasal infection did not develop any symptoms. This is because activated responses in the nasal cavity significantly suppressed virus production. Another word is this nasal vaccine completely protected the mice from disease development and mortality. Therefore, the bivalent RBD vaccine platform has potential to be developed into an anti-SARS-CoV-2 universal vaccine.

Higher correlation between neutralizing antibodies and surrogate neutralizing or binding antibodies in COVID-19 patients than vaccine recipients.

This study determined the seropositive rates and levels of antibodies to severe acute respiratory syndrome coronavirus-2 in 50 patients and 108 vaccinees using microneutralization test (MNT), surrogate virus neutralization test (sVNT), chemiluminescent microparticle immunoassay (CMIA), and electrochemiluminescence immunoassay (ECLIA). MNT, as the reference method, employed living clade S and Delta viruses to measure neutralizing (NT) antibodies, while sVNT employed wild type strain and Delta receptor-binding domains (RBD) as the test antigens to measure sVNT antibodies. CMIA and ECLIA employed only one version of RBD to measure the binding antibodies. Our study performed S gene sequencing of the test virus to exclude undesired mutants that might lead to changes in antibody levels in MNT assay. We showed that spike protein amino acid sequences of our Delta virus contained 13 amino acid changes, with 3 related to the reduced neutralization. The MNT assay showed a significant reduction in seropositive rates and antibody levels in the patients' sera when the Delta variant replaced clade S as the test virus. In contrast, the seropositive rates determined by sVNT assay using wild type strain RBD and Delta RBD were non-significantly different, suggesting that sVNT assay could not identify the difference between the antigenicity of wild type RBD and Delta RBD. Furthermore, the correlation between the levels of NT and sVNT antibodies was moderate with the patients' sera but modest with the post-vaccination sera. The seropositive rates in the patients, as determined by CMIA or ECLIA, were not different from the MNT assay using clade S, but not Delta, as the test virus. In all analyses, the correlations between the antibody levels measured by MNT and the other 3 assays were modest to moderate, with the r-values of 0.3500-0.7882.

A novel film spray containing curcumin inhibits SARS-CoV-2 and influenza virus infection and enhances mucosal immunity.

BACKGROUND: Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza virus is still a major worldwide health concern. Plants are a good source of bioactive compounds to be used as preventive measures for both inhibiting the virus binding and enhancing mucosal innate immunity. Curcumin has been shown to possess antiviral activity and modulate innate immunity. Therefore, the purpose of this study was to develop an oro-nasal film spray containing curcumin and determine its antiviral activity against SARS-CoV-2 and influenza virus infection, as well as its effects on mucosal innate immunity and inflammatory cytokines in vitro. METHODS: The antiviral activity of the film spray against SARS-CoV-2, influenza A/H1N1, A/H3N2, and influenza B was assessed in vitro by plaque reduction assay. Cytotoxicity of the film spray to oral keratinocytes and nasal epithelial cells was assessed by MTT assay, and cytotoxicity to Vero and MDCK cells was assessed by an MTS-based cytotoxicity assay. Oral and nasal innate immune markers in response to the film spray were determined by ELISA and by a commercial Milliplex Map Kit, respectively. RESULTS: Our data show that the film spray containing curcumin can inhibit both SARS-CoV-2 and influenza virus infections while maintaining cell viability. Results obtained among 4 viruses revealed that curcumin film spray demonstrated the highest inhibitory activity against SARS-CoV-2 with the lowest EC50 of 3.15 µg/ml and the highest SI value of 4.62, followed by influenza B (EC50 = 6.32 µg/ml, SI = 2.04), influenza A/H1N1 (EC50 = 7.24 µg/ml, SI = 1.78), and influenza A/H3N2 (EC50 > 12.5 µg/ml, SI < 1.03), respectively. Antimicrobial peptides LL-37 and HD-5, IL-6 and TNF-α produced by oral keratinocytes were significantly induced by the film spray, while hBD2 was significantly reduced. CONCLUSION: Film spray containing curcumin possesses multiple actions against SARS-CoV-2 infection by inhibiting ACE-2 binding in target cells and enhancing mucosal innate immunity. The film spray can also inhibit influenza virus infection. Therefore, the curcumin film spray may be effective in preventing the viral infection of both SARS-CoV-2 and influenza.

Safety and immunogenicity of locally produced trivalent inactivated influenza vaccine (Tri Fluvac) in healthy Thai adults aged 18-64 years in Nakhon Phanom: A Phase III double blinded, three-arm, randomized, controlled trial.

BACKGROUND: Domestic influenza vaccine production facilitates a sustainable supply for mitigating seasonal influenza and improves national health security by providing infrastructure and experience for pandemic vaccine production, if needed. METHODS: A Phase III, double blind, randomized controlled trial was conducted from Sep 2019-Oct 2020 in healthy adults 18-64 years in Nakhon Phanom, Thailand. Randomization (3:3:1) compared study vaccine (Tri Fluvac), saline placebo, and an active comparator (licensed vaccine). Primary outcomes were superior efficacy compared to placebo based on RT-PCR-confirmed influenza virus infection within 12 months and non-inferiority compared to active comparator based on immunogenicity (HAI assay) at 28 days. Safety was also assessed. RESULTS: The trial enrolled 4,284 participants (Tri Fluvac = 1,836; placebo = 1,836; active comparator = 612). There were 29 RT-PCR positive influenza infections (10 Tri Fluvac, 5.5/1,000 PY; 19 placebo, 10.4/1,000PY; 0 comparator) for an absolute protective efficacy of 46.4 (95 % CI = -22.0-76.5) compared with placebo, but the power was 43.7 %. Seroconversion difference rates between Tri Fluvac and comparator at Day 28 were 1.74 (95 % CI: -2.77, 6.25), 2.22 (-2.40, 6.84), and -0.57 (-5.41, 4.27) for A(H1N1), A(H3N2), and B strains, respectively. Adverse and severe adverse events occurred in 175 (9.5 %) Tri Fluvac, 177 (10.8 %) placebo, and 66 (10.8 %) comparator arms (p-value = 0.437, Tri Fluvac vs. comparator) CONCLUSIONS: Tri Fluvac was well tolerated, and immunogenicity was non-inferior to the active comparator, meeting U.S. Food and Drug Administration (FDA) criteria for adult vaccine licensure. Few acute respiratory infections were reported during intense COVID-19 pandemic restrictions, resulting in insufficient power to evaluate clinical efficacy.

Phenotypic and functional changes of T cell subsets after CoronaVac vaccination.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection. METHODS: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot). FINDINGS: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4+ T cell-, but not CD8+ T cell-responses. Among the CD4+ T cells, CoronaVac activated mainly Th2 (CD4+ T) cells. Serum antibody levels significantly declined three months after the second dose. INTERPRETATION: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection.

Seroprevalence of anti-SARS-CoV-2 antibodies in Thai adults during the first three epidemic waves.

This study determined the presence of anti-SARS-CoV-2 antibodies in 4964 individuals, comprising 300 coronavirus disease-19 (COVID-19) prepandemic serum samples, 142 COVID-19 patients, 2113 individuals at risk due to their occupations, 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, and 553 Thai citizens returning after spending extended periods of time in countries with a high disease prevalence. We recruited participants between May 2020 and May 2021, which spanned the first two epidemic waves and part of the third wave of the COVID-19 outbreaks in Thailand. Their sera were tested in a microneutralization and a chemiluminescence immunoassay for IgG against the N protein. Furthermore, we performed an immunofluorescence assay to resolve discordant results between the two assays. None of the prepandemic sera contained anti-SARS-CoV-2 antibodies, while antibodies developed in 88% (15 of 17) of the COVID-19 patients at 8-14 days and in 94-100% of the patients between 15 and 60 days after disease onset. Neutralizing antibodies persisted for at least 8 months, longer than IgG antibodies. Of the 2113 individuals at risk due to their occupation, none of the health providers, airport officers, or public transport drivers were seropositive, while antibodies were present in 0.44% of entertainment workers. Among the 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, seropositivity was present in 1.9, 1.5, and 7.5% of the Bangkok residents during the three epidemic waves, respectively, and in 1.3% of the Chiang Mai people during the first epidemic wave. The antibody prevalence varied between 6.5 and 47.0% in 553 Thai people returning from high-risk countries. This serosurveillance study found a low infection rate of SARS-CoV-2 in Thailand before the emergence of the Delta variant in late May 2021. The findings support the Ministry of Public Health's data, which are based on numbers of patients and contact tracing.

Antiviral effect in association with anti-apoptosis and anti-autophagy of repurposing formoterol fumarate dihydrate on enterovirus A71-infected neuronal cells.

OBJECTIVE: To investigate antiviral activity, anti-apoptosis and anti-autophagy associated with antiviral effect of repurposing formoterol fumarate dihydrate (FFD) against enterovirus A71 (EV-A71) infection in human neuroblastoma cells. METHODS: In vitro antiviral effects of FFD against EV-A71 infection were examined in human neuroblastoma SK-N-SH cells. The impacts on EV-A71 replication were evaluated by progeny virus production, viral RNA synthesis, and viral protein expression. The target of action of FFD against EV-A71 was determined from the effective stage by time-of-addition assay. Moreover, the anti-apoptosis and anti-autophagy activities associated with antiviral effect were observed by detection of apoptosis- and autophagy-related proteins. RESULTS: FFD significantly inhibited EV-A71 replication in neuronal cells through interfering the early stages of replication cycle which might be the steps during uncoating to viral protein synthesis. Additionally, FFD culminated in reducing of EV-A71-induced apoptosis and autophagy with caspase-3-cleaved form and LC3-II expression levels showed markedly decreased while increasing of Bcl-2 and mTOR expression levels. These might indicate the neuroprotective effect of FFD on EV-A71-induced apoptosis and autophagy. CONCLUSIONS: Preliminary mode of action studies showed that repurposing FFD significantly inhibited EV-A71 replication at early stage of viral replication and exhibited anti-apoptosis and anti-autophagy activities in neuronal cells. These findings may provide an opportunity, via drug repurposing of FFD, for a candidate antiviral drug against EV-A71 infection.

Evaluation of different platforms for the detection of anti-SARS coronavirus-2 antibodies, Thailand.

BACKGROUND: Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) help determine previous infection in individuals, regardless of whether they are asymptomatic or symptomatic. The detection of antibodies serves several purposes, including supporting other assays for disease diagnosis, conducting seroepidemiological studies, and evaluating vaccines. Many platforms of immunological methods for anti-SARS-CoV-2 antibody detection and their performance require validation. METHODS: This study evaluated the test performance of three autoanalyzer-based assays (Architect IgG, Vitros IgG, and Vitros total Ig) and one manual ELISA (Wantai total Ig) against a microneutralization (microNT) assay on the detection of SARS-CoV-2 antibodies. Furthermore, an indirect immunofluorescence assay verified the discordant results between the microNT and commercial assays. The test sensitivity, specificity, positive predictive value, and negative predictive value were determined based on four groups of 1005 serum samples: 102 COVID-19 prepandemic sera, 45 anti-SARS-CoV-2 positive sera, 366 sera of people at risk, and 492 sera of citizens returning from countries with a high prevalence of infection. RESULTS: The analyses as a whole showed that the performance of these commercial assays was comparable. Each group was also analysed separately to gain further insight into test performance. The Architect did not detect two positive sera of people at risk (prevalence of infection 0.55%). The other methods correctly identified these two positive sera but yielded varying false-positive results. The group of returning travellers with an infection rate of 28.3% (139 of 492) better differentiated the test performance of individual assays. CONCLUSIONS: High-throughput Architect and Vitros autoanalyzers appear appropriate for working on large sample sizes in countries that can afford the cost. The Wantai ELISA, while requiring more individual time and technical skill, may provide reliable results at a lower cost. The selection of assays will depend on the laboratory facilities and feasibility.

Response of Severe EV71-Infected Patients to Hyperimmune Plasma Treatment: A Pilot Study.

Hand, foot, and mouth disease (HFMD) is highly prevalent in East and Southeast Asia. It particularly affects children under five years of age. The most common causative agents are coxsackieviruses A6 and A16, and enterovirus A71 (EV71). The clinical presentation is usually mild and self-limited, but, in some cases, severe and fatal complications develop. To date, no specific therapy or worldwide vaccine is available. In general, viral infection invokes both antibody and cell-mediated immune responses. Passive immunity transfer can ameliorate the severe symptoms of diseases such as COVID-19, influenza, MERS, and SARS. Hyperimmune plasma (HIP) from healthy donors with high anti-EV71 neutralizing titer were used to transfuse confirmed EV71-infected children with neurological involvement (n = 6). It resulted in recovery within three days, with no neurological sequelae apparent upon examination 14 days later. Following HIP treatment, plasma chemokines were decreased, whereas anti-inflammatory and pro-inflammatory cytokines gradually increased. Interestingly, IL-6 and G-CSF levels in cerebrospinal fluid declined sharply within three days. These findings indicate that HIP has therapeutic potential for HFMD with neurological complications. However, given the small number of patients who have been treated, a larger cohort study should be undertaken. Successful outcomes would stimulate the development of anti-EV71 monoclonal antibody therapy.

Molecular Characterization of Seasonal Influenza A and B from Hospitalized Patients in Thailand in 2018-2019.

Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites (S91R, S181T, T202I) as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission.

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

BACKGROUND: Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising. METHODS: This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN-γ ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection. RESULTS: IFN-γ ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN-γ ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN-γ and TNF-α, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry. CONCLUSIONS: This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.

Elevation of Cleaved p18 Bax Levels Associated with the Kinetics of Neuronal Cell Death during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

Seroprevalence of antibodies to enterovirus 71 and coxsackievirus A16 among people of various age groups in a northeast province of Thailand.

BACKGROUND: Hand, foot and mouth disease (HFMD) is endemic among population of young children in Thailand. The disease is mostly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16). METHODS: This study conducted serosurveillance for neutralizing (NT) antibodies to EV71 subgenotypes B5 and C4a, and to CA16 subgenotypes B1a and B1b, in 579 subjects of various ages using a microneutralization assay in human rhabdomyosarcoma (RD) cells. These test viruses were the major circulating subgenotypes associated with HFMD in Thailand during the study period. RESULTS: We found that the levels of seropositivity against all 4 study viruses were lowest in the age group of 6-11 months, i.e., 5.5% had antibody to both EV71 subgenotypes, while 14.5% and 16.4% had antibody to CA16 subgenotypes B1a and B1b, respectively. The percentages of subjects with antibodies to these 4 viruses gradually increased with age, but were still less than 50% in children younger than 3 years. These laboratory data were consistent with the epidemiological data collected by the Ministry of Public Health which showed repeatedly that the highest number of HFMD cases was in children aged 1 year. Analyses of amino acid sequences of the test viruses showed 97% identity between the two subgenotypes of EV71, and 99% between the two subgenotypes of CA16. Nevertheless, the levels of seropositivity and antibody titer against the two subgenotypes of EV71 and of CA16 were not significantly different. CONCLUSIONS: This study clearly demonstrated NT antibody activity across EV71-B5 and EV71-C4a subgenotypes, and also across CA16-B1a and CA16-B1b subgenotypes. Moreover, there were no significant differences by gender in the seropositive rates and antibody levels to any of the 4 virus subgenotypes.

The Effect of Detectable HIV Viral Load among HIV-Infected Children during Antiretroviral Treatment: A Cross-Sectional Study.

The RNA viral load of human immunodeficiency virus (HIV) is initially used to determine the status of the HIV infection. The goal of therapy following treatment failure is to achieve and maintain virologic suppression. A detectable viral load may relate to the progression of HIV infection. A cross-sectional survey was conducted from January 2013 to December 2014 at the Bamrasnaradura Infectious Diseases Institute, Thailand. The aim was to determine the prevalence of detectable HIV viral load (dVL) and analyze the factors associated with post-dVL conditions that occur independently of a switch to a new antiretroviral agent. The prevalence of dVL was 27% (27 of 101). The mean ages of dVL and non-dVL children were 12.0 and 12.3 years, respectively. Age, sex, body mass index for age z-scores, previous tuberculosis disease history and parental tuberculosis history of both groups were not significantly different (p > 0.05). The prevalence of poor adherence (<95%), influenza-like illness (ILI) and opportunistic infections were higher in dVL than non-dVL children (p < 0.05). The mean nadir CD4 cell count during the study was lower in dVL than non-dVL children (646 compared to 867, respectively; p < 0.05). Other factors were not significant (all p > 0.05). In multivariable analysis, dVL was significantly associated with ILI (odds ratio (OR) = 9.6, 95% confidence interval (CI) = 1.3-69.4), adherence (OR = 0.195, 95% CI = 0.047-0.811) and nadir CD4 during the study (OR = 1.102, 95% CI = 1.100-1.305). The prevalence of dVL was 27% with this dVL among HIV-infected children found to be associated with ILI, poor adherence and lower nadir CD4 during the study.

Kinetics, Longevity, and Cross-Reactivity of Antineuraminidase Antibody after Natural Infection with Influenza A Viruses.

The kinetics, longevity, and breadth of antibodies to influenza virus neuraminidase (NA) in archival, sequential serum/plasma samples from influenza A virus (IAV) H5N1 infection survivors and from patients infected with the 2009 pandemic IAV (H1N1) virus were determined using an enzyme-linked lectin-based assay. The reverse-genetics-derived H4N1 viruses harboring a hemagglutinin (HA) segment from A/duck/Shan Tou/461/2000 (H4N9) and an NA segment derived from either IAV H5N1 clade 1, IAV H5N1 clade 2.3.4, the 2009 pandemic IAV (H1N1) (H1N1pdm), or A/Puerto Rico/8/1934 (H1N1) virus were used as the test antigens. These serum/plasma samples were also investigated by microneutralization (MN) and/or hemagglutination inhibition (HI) assays. Neuraminidase-inhibiting (NI) antibodies against N1 NA of both homologous and heterologous viruses were observed in H5N1 survivors and H1N1pdm patients. H5N1 survivors who were never exposed to H1N1pdm virus developed NI antibodies to H1N1pdm NA. Seroconversion of NI antibodies was observed in 65% of the H1N1pdm patients at day 7 after disease onset, but an increase in titer was not observed in serum samples obtained late in infection. On the other hand, an increase in seroconversion rate with the HI assay was observed in the follow-up series of sera obtained on days 7, 14, 28, and 90 after infection. The study also showed that NI antibodies are broadly reactive, while MN and HI antibodies are more strain specific.

Safety and immunogenicity of a live attenuated influenza H5 candidate vaccine strain A/17/turkey/Turkey/05/133 H5N2 and its priming effects for potential pre-pandemic use: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: The emergence of highly pathogenic avian influenza H5N1 viruses has raised concerns about their pandemic potential. Vaccination is the most effective way of preventing influenza. In this study, we investigated the safety and immunogenicity of an avian H5N2 live attenuated influenza vaccine (LAIV H5N2) in healthy Thai adults and its priming immune responses with an H5N1 inactivated vaccine boost. METHODS: This study was done at the Vaccine Trial Centre at Mahidol University, Bangkok, Thailand and was divided into two parts. Part 1 consisted of a randomised, double-blind, placebo-controlled trial done over 18 months. We randomly assigned (2:1) healthy Thai adults aged 18-49 years with a computer generated randomisation sequence (blocks of six) to receive either two intranasal doses (0·25 mL per nostril) of LAIV H5N2 (101 participants) or placebo (51 participants) 21 days apart. For part 2, an open-label trial was done in which previously vaccinated participants (40 from LAIV H5N2 group and 20 placebo) were given one intramuscular dose (0·5 mL) of H5N1 booster vaccine. Participants, investigators, and site-study workers were blinded from randomisation. Immune responses after subsequent immunisation were evaluated using haemagglutination-inhibition and microneutralisation assays and circulating follicular T-helper cells and plasmablast cells were measured in serum and whole blood. The trials are registered with ClinicalTrials.gov, numbers NCT01841918 and NCT02229357. FINDINGS: Between Feb 4, 2013, and Feb 28, 2013, 256 individuals were screened, of whom 152 participants were enrolled in part 1 of this study. LAIV H5N2 vaccine was well tolerated. Viral shedding was detected in only six (6%) of 101 participants in the vaccine group 1 day after the first vaccination and in and two (2%) of 98 participants in the group after the second vaccination. There was no serious adverse event in both groups. 51 (50%) of 101 participants in the vaccine group and 28 (55%) of 51 in the placebo group reported at least one adverse event. 80 (84%) of 95 events in the vaccine group and 32 (78%) of 43 events in the placebo groups were reportedly suspected adverse events, probably related to the vaccine; however, most were mild in nature. After two doses of vaccine, 13 (13%) of 100 participants in the vaccine group had an increase in haemagglutination-inhibition titre of more than four-fold and four (4%) of 100 vaccinees developed a rise in neutralisng antibody titre of more than four-fold. 1 year later, after a booster with an inactivated H5N1 vaccine (part 2), 39 (98%) of 40 participants who had previously been vaccinated with LAIV H5N2 had an increase in haemagglutination-inhibition titre of greater than four-fold as early as day 7 compared with three (15%) of 20 participants in the placebo group. Peak geometric mean titre (GMT) for haemagglutination-inhibition antibodies in the previously LAIV H5N2 vaccinated group (566·89 [95% CI 436·97-735·44]) were significantly higher than among those who previously received placebo (25·49 [11·82-54·96]; p<0·0001). The peak GMT by neutralising antibody assay in the H5N2 vaccinated group (1395·85 [1040·79-1872·03]) was also significantly higher than that observed in the placebo group (17·41 [9·05-33·48]; p<0·0001). Importantly, higher cross-reactive haemagglutination-inhibition antibody titres against H5N1 (clades 1, 2.1.3.2, and 2.3.4) were detected in the LAIV H5N2 experienced group than the naive group (p<0·0001). INTERPRETATION: Our data suggest that LAIV vaccination induces long-lasting memory immune responses. The limitation of this study was that part 2 was designed as a proof-of-concept study by contrast with part 1. FUNDING: WHO.

Cross-reactive antibodies against H7N9 and H5N1 avian influenza viruses in Thai population.

BACKGROUD: Avian influenza H5N1 and H7N9 viruses have jumped across species from avian to humans and become a threat to public health. Not much is known about pre-existing cross-reactive antibodies against these avian viruses in human population. OBJECTIVE: To determine the prevalence of cross-reactive anti-HA and anti-NA antibodies to avian influenza H5N1 and H7N9 viruses in Thai population. METHOD: Archival serum samples from 100 blood donors and 21 patients infected with 2009 pandemic influenza A (H1N1) (pdmH1N1) virus were investigated by hemagglutination-inhibition (HAI) and neuraminidase-inhibition (NAI) assays for anti-HA and anti-NA antibodies, respectively. The test antigens comprised 2 human viruses (pdmH1N1 and H3N2 viruses), and 6 reassortant viruses carrying HA and NA genes of avian H5N1 or H7N9 virus generated by reverse genetics. RESULTS: HAI antibody titers ≥ 10 were found in 58, 89, 0 and 15% of blood donors as tested against pdmH1N1, H3N2, H5N1 and H7N9 viruses, respectively. On the other hand, NAI antibodies were detected in 98, 94, 73 and 53% of blood donors when reverse genetic-derived viruses harboring NA gene from pdmH1N1, H3N2, H5N1 or H7N9 virus were used as the test antigens. Moreover, 66.7% of pdmH1N1 patients who had > 4 fold increase in HAI antibody titers in paired sera developed > 4 fold increase in NAI antibody titers. CONCLUSIONS: Anti-NA antibody has broader reactivity than anti-HA antibody, therefore, it can be a supplement to anti-HA antibody in the prevention against novel influenza viruses.

Immunobiological properties of influenza A (H7N9) hemagglutinin and neuraminidase proteins.

Recombinant vaccinia viruses harboring the complete hemagglutinin (HA) or neuraminidase (NA) genes from the influenza A/Anhui/1/2013 (H7N9) virus were constructed (rVac-H7 HA and rVac-N9 NA viruses). The HA and NA proteins were expressed in the cytoplasm and on the plasma membrane of thymidine-kinase-negative (TK(-)) cells infected with these recombinant viruses. Only one form of the HA protein was expressed in infected TK(-) cells, with a molecular weight (MW) of 75 kDa, but three forms were found when the culture medium was supplemented with trypsin (MWs of 75, 50 and 27 kDa), which was similar to what was found in Madin-Darby canine kidney (MDCK) cells infected with reverse genetic (rg) influenza viruses carrying HA genes of H7N9 virus origin. One form of hyperglycosylated NA protein with a MW of 75 kDa was produced in rVac-N9-NA-virus-infected TK(-) or MDCK cells. The MW decreased to 55 kDa after deglycosylation. The hyperglycosylated recombinant NA protein demonstrated sialidase activity in a fetuin-based neuraminidase assay. The rVac-H7 HA and rVac-N9 NA viruses elicited significantly higher anti-HA and anti-NA antibody titers in BALB/c mice that were immunized once than in ICR mice. The anti-HA and anti-NA antibodies showed activity against homosubtypic HA or NA, but not against heterosubtypic HA or NA, as determined by hemagglutination-inhibition and microneutralization assays for anti-HA antibodies and neuraminidase-inhibition and replication-inhibition assays for anti-NA antibodies. Taken together, our data demonstrated immunobiological properties of recombinant HA and NA proteins that might be useful for vaccine development.

Lack of transmission among healthcare workers in contact with a case of Middle East respiratory syndrome coronavirus infection in Thailand.

INTRODUCTION: A hospital-associated outbreak of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was reported. We aimed to assess the effectiveness of infection control measures among healthcare workers (HCWs) who were exposed to a MERS patient and/or his body fluids in our institute. METHODS: A descriptive study was conducted among HCWs who worked with a MERS patient in Bamrasnaradura Infectious Diseases Institute, Thailand, between 18 June and 3 July 2015. Contacts were defined as HCWs who worked in the patient's room or with the patient's body fluids. Serum samples from all contacts were collected within 14 days of last contact and one month later. Paired sera were tested for detection of MERS-CoV antibodies by using an indirect ELISA. RESULTS: Thirty-eight (88.4 %) of 43 identified contacts consented to enroll. The mean (SD) age was 38.1 (11.1) years, and 79 % were females. The median (IQR) cumulative duration of work of HCWs in the patient's room was 35 (20-165) minutes. The median (IQR) cumulative duration of work of HCWs with the patient's blood or body fluids in laboratory was 67.5 (43.7-117.5) minutes. All contacts reported 100 % compliance with hand hygiene, using N95 respirator, performing respirator fit test, wearing gown, gloves, eye protection, and cap during their entire working period. All serum specimens of contacts tested for MERS-CoV antibodies were negative. CONCLUSIONS: We provide evidence of effective infection control practices against MERS-CoV transmission in a healthcare facility. Strict infection control precautions can protect HCWs. The optimal infection control measures for MERS-CoV should be further evaluated.