Multiple SARS-CoV-2 immunizations of an unvaccinated population lead to complex immunity. A T cell reactivity study of blood donors in Antananarivo.

BACKGROUND: Madagascar has undergone multiple and robust COVID-19 waves. The resulting immune background developed by its poorly vaccinated population has however not been described. METHODS: In this study, serological analysis and specific T cell response descriptions were used to describe the history of exposures of the capital's blood donors to SARS-CoV-2 and its VOCs. Samples were collected early 2022, and pools of multiple immunogenic peptides of SARS-CoV-2 were used in an IFN-γ secretion ELISPOT assay to characterize the specific T-cell immunity developed against these potential epitopes. RESULTS: Multiple epidemic waves have led to 92.1% of donors having detectable antibodies, and 94.8% having developed T-cells against SARS-CoV-2. Heterogeneous reactivities to different strain-derived peptides suggested multiple immunological backgrounds in the population including 16.1% of individuals exposed at least once to a unique strain, 27.1% to two strains, 28.5% to three strains, and 23.1% to four distinct strains. CONCLUSIONS: Cross-reactivity increased with multiple exposures but did not decrease the risk of re-infection. These results describe the extremely complex immunological background developed following multiple natural immunizations.

Epidemiology and pathological progression of erythematous lip lesions in captive sun bears (Helarctos malayanus).

This study investigates the occurrence of erythematous lip lesions in a captive sun bear population in Cambodia, including the progression of cheilitis to squamous cell carcinoma, and the presence of Ursid gammaherpesvirus 1. Visual assessment conducted in 2015 and 2016 recorded the prevalence and severity of lesions. Opportunistic sampling for disease testing was conducted on a subset of 39 sun bears, with histopathological examination of lip and tongue biopsies and PCR testing of oral swabs and tissue biopsies collected during health examinations. Lip lesions were similarly prevalent in 2015 (66.0%) and 2016 (68.3%). Degradation of lip lesion severity was seen between 2015 and 2016, and the odds of having lip lesions, having more severe lip lesions, and having lip lesion degradation over time, all increased with age. Cheilitis was found in all lip lesion biopsies, with histological confirmation of squamous cell carcinoma in 64.5% of cases. Single biopsies frequently showed progression from dysplasia to neoplasia. Eighteen of 31 sun bears (58.1%) had at least one sample positive for Ursid gammaherpesvirus 1. The virus was detected in sun bears with and without lip lesions, however due to case selection being strongly biased towards those showing lip lesions it was not possible to test for association between Ursid gammaherpesvirus 1 and lip squamous cell carcinoma. Given gammaherpesviruses can play a role in cancer development under certain conditions in other species, we believe further investigation into Ursid gammaherpesvirus 1 as one of a number of possible co-factors in the progression of lip lesions to squamous cell carcinoma is warranted. This study highlights the progressively neoplastic nature of this lip lesion syndrome in sun bears which has consequences for captive and re-release management. Similarly, the detection of Ursid gammaherpesvirus 1 should be considered in pre-release risk analyses, at least until data is available on the prevalence of the virus in wild sun bears.

Impaired Antibody-Independent Immune Response of B Cells in Patients With Acute Dengue Infection.

Dengue is a mosquito-borne viral disease caused by dengue virus (DENV). The disease is endemic to more than 100 countries with 390 million dengue infections per year. Humoral immune responses during primary and secondary DENV infections are well-investigated. However, the impact of DENV infection on B cell subsets and their antibody-independent functions are not well-documented. Through this study, we aimed to define the distribution of B cell subsets in the acute phase of DENV infection and characterize the effect of DENV infection on B cell functions such as differentiation into memory and plasma cells and cytokine production. In our cohort of Cambodian children, we observed decreased percentages of CD24hiCD38hi B cells and CD27- naïve B cells within the CD19 population and increased percentages of CD27+CD38hiCD138+ plasma cells as early as 4 days post appearance of fever in patients with severe dengue compared to patients with mild disease. Lower percentages of CD19+CD24hiCD38hi B cells in DENV-infected patients were associated with decreased concentrations of soluble CD40L in patient plasma and decreased platelet counts in these patients. In addition, CD19+CD24hiCD38hi and CD19+CD27- B cells from DENV-infected patients did not produce IL-10 or TNF-α upon stimulation in vitro, suggesting their contribution to an altered immune response during DENV infection. In addition, CD19+CD27- naïve B cells isolated from dengue patients were refractory to TLR/anti-IgM stimulation in vitro, which correlated to the increased expression of inhibitory Fcγ receptors (FcγR) CD32 and LILRB1 on CD19+CD27- naïve B cells from DENV-infected patients. Collectively, our results indicate that a defective B cell response in dengue patients may contribute to the pathogenesis of dengue during the early phase of infection.

Asymptomatic Dengue Virus Infections, Cambodia, 2012-2013.

We investigated dengue virus (DENV) and asymptomatic DENV infections in rural villages of Kampong Cham Province, Cambodia, during 2012 and 2013. We conducted perifocal investigations in and around households for 149 DENV index cases identified through hospital and village surveillance. We tested participants 0.5-30 years of age by using nonstructural 1 rapid tests and confirmed DENV infections using quantitative reverse transcription PCR or nonstructural 1-capture ELISA. We used multivariable Poisson regressions to explore links between participants' DENV infection status and household characteristics. Of 7,960 study participants, 346 (4.4%) were infected with DENV, among whom 302 (87.3%) were <15 years of age and 225 (65.0%) were <9 years of age. We identified 26 (7.5%) participants with strictly asymptomatic DENV infection at diagnosis and during follow-up. We linked symptomatic DENV infection status to familial relationships with index cases. During the 2-year study, we saw fewer asymptomatic DENV infections than expected based on the literature.

Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

Cross-reactivities between human IgMs and the four serotypes of dengue virus as probed with artificial homodimers of domain-III from the envelope proteins.

BACKGROUND: Dengue fever is the most important vector-borne viral disease. Four serotypes of dengue virus, DENV1 to DENV4, coexist. Infection by one serotype elicits long-lasting immunity to that serotype but not the other three. Subsequent infection by a different serotype is a risk factor for severe dengue. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by neutralizing antibodies. We determined the serotype specificity and cross-reactivity of human IgMs directed against ED3 by using a well-characterized collection of 90 DENV-infected and 89 DENV-uninfected human serums. METHODS: The recognitions between the four serotypes of ED3 and the serums were assayed with an IgM antibody-capture ELISA (MAC-ELISA) and artificial homodimeric antigens. The results were analyzed with Receiving Operator Characteristic (ROC) curves. RESULTS: The DENV-infected serums contained IgMs that reacted with one or several ED3 serotypes. The discrimination by ED3 between serums infected by the homotypic DENV and uninfected serums varied with the serotype in the decreasing order DENV1 > DENV2 > DENV3 > DENV4. The ED3 domain of DENV1 gave the highest discrimination between DENV-infected and DENV-uninfected serums, whatever the infecting serotype, and thus behaved like a universal ED3 domain for the detection of IgMs against DENV. Some ED3 serotypes discriminated between IgMs directed against the homotypic and heterotypic DENVs. The patterns of cross-reactivities and discriminations varied with the serotype. CONCLUSIONS: The results should help better understand the IgM immune response and protection against DENV since ED3 is widely used as an antigen in diagnostic assays and an immunogen in vaccine candidates.

Thermodynamic stability of domain III from the envelope protein of flaviviruses and its improvement by molecular design.

The Flavivirus genus includes widespread and severe human pathogens like the four serotypes of dengue virus (DENV1 to DENV4), yellow fever virus, Japanese encephalitis virus and West Nile virus. Domain III (ED3) of the viral envelope protein interacts with cell receptors and contains epitopes recognized by virus neutralizing antibodies. Its structural, antigenic and immunogenic properties have been thoroughly studied contrary to its physico-chemical properties. Here, the ED3 domains of the above pathogenic flaviviruses were produced in the periplasm of Escherichia coli. Their thermodynamic stabilities were measured and compared in experiments of unfolding equilibriums, induced with chemicals or heat and monitored through protein fluorescence. A designed ED3 domain, with the consensus sequence of DENV strains from all serotypes, was highly stable. The low stability of the ED3 domain from DENV3 was increased by three changes of residues in the protein core without affecting its reactivity towards DENV-infected human serums. Additional changes showed that the stability of ED3 varied with the DENV3 genotype. The T(m) of ED3 was higher than 69°C for all the tested viruses and reached 86°C for the consensus ED3. The latter, deprived of its disulfide bond by mutations, was predominantly unfolded at 20°C. These results will help better understand and design the properties of ED3 for its use as diagnostic, vaccine or therapeutic tools.

Hospitalized cases of influenza A(H1N1)pdm09 in the French territories of the Americas, July 2009–March 2010 / Casos hospitalizados de gripe A(H1N1)pdm09 en los territorios franceses de las Américas entre julio de 2009 y marzo de 2010

Objective. To describe the methodology used for implementing a surveillance system specifically for influenza A(H1N1)pdm09 in the French West Indies and French Guiana during an outbreak of this new virus in 2009­2010, and to report its main results. Methods. This was an observational descriptive study of confirmed and probable cases of influenza A(H1N1)pdm09 hospitalized for at least 24 hours in 23 July 2009­3 March 2010. Reverse transcription polymerase chain reaction was performed on nasopharyngeal swab samples according to the Centers for Disease Control and Prevention protocol. A probable case was defined as fever ≥ 38°C or aches or asthenia with respiratory symptoms (cough or dyspnea). All confirmed and probable hospitalized cases were reported, along with patient's age, sex, clinical condition at admission, place and length of hospitalization, antiviral treatment, underlying conditions, complications, and clinical evolution. A case was classified as severe if respiratory assistance or intensive care was required or if death resulted. Results. A total of 331 confirmed and 16 probable cases were hospitalized, with a hospitalization rate ranging from 4.3 per 1 000 clinical cases in Saint Martin to 10.3 in French Guiana. Of these, 36 were severe, and subsequently, 10 were fatal. The median length of stay was 4 days for non-severe cases and 9 days for severe (P < 0.05). The mean patient age was 21 years, and severe cases were significantly older than non-severe (mean: 38 years versus 19 years, P < 0.05). Underlying conditions associated with a higher risk of severity were 65 years of age or more (RR = 7.5, 95%CI = 4.2­13.3), diabetes (RR = 3.7, 95%CI = 1.5­9.4), cardiac insufficiency (RR = 8.4, 95%CI = 5.2­13.6), and morbid obesity (RR = 4.4, 95%CI = 1.8­ 10.4). Patients who received antiviral treatment within 2 days of symptom onset had shorter hospital stays (mean: 4 days versus 6.5 days, P < 0.05), and the illness tended to become less severe (11.1% versus 19.0%, P = 0.13). Conclusions. Active research of hospitalized cases enabled almost exhaustive surveillance. The pandemic's hospitalization rates and lethality were more moderate than expected. Some previously known underlying conditions of severity were confirmed dur.

Objetivo. Describir la metodología usada para implementar un sistema de vigilancia específico para la gripe A(H1N1)pdm09 en las Indias Occidentales Francesas y la Guayana Francesa durante un brote ocasionado por este virus nuevo ocurrido en 20092010 y presentar sus principales resultados. Métodos. Se llevó a cabo un estudio de observación descriptivo de los casos confirmados y probables de gripe por A(H1N1)pdm09 hospitalizados durante al menos 24 horas entre el 23 de julio de 2009 y el 3 de marzo de 2010. De conformidad con el protocolo de los Centros para el Control y la Prevención de Enfermedades se realizó la prueba de reacción en cadena de la polimerasa con transcripción inversa en muestras de hisopados nasofaríngeos. Se definió como caso probable la presencia de fiebre ≥ 38 °C o dolores o astenia junto con síntomas respiratorios (tos o disnea). Se comunicaron todos los casos hospitalizados confirmados y probables junto con la edad, el sexo, la situación clínica del paciente en el momento del ingreso, el lugar y la duración de la hospitalización, el tratamiento antivírico, las enfermedades subyacentes, las complicaciones y la evolución clínica. Se clasificaron como graves los casos que requirieron asistencia respiratoria o cuidados intensivos o provocaron la muerte. Resultados. Fueron hospitalizados en total 331 casos confirmados y 16 probables, con una tasa de hospitalización que osciló entre 4,3 por cada 1 000 casos clínicos en San Martín y 10,3 por cada 1 000 en la Guayana Francesa. De ellos, 36 fueron graves y 10 llevaron posteriormente a la muerte del paciente. La mediana de la duración de las hospitalizaciones fue de 4 días para los casos no graves y de 9 días para los graves (P < 0,05). La edad media de los pacientes fue de 21 años, y los casos graves fueron significativamente de mayor edad que los no graves (media: 38 años frente a 19 años; P < 0,05). Las enfermedades subyacentes asociadas con un riesgo mayor de gravedad fueron edad de 65 años o más (RR = 7,5; IC de 95% = 4,213,3), diabetes (RR = 3,7; IC de 95% = 1,59,4), insuficiencia cardíaca (RR = 8,4; IC de 95% = 5,213,6) y obesidad mórbida (RR = 4,4; IC de 95% = 1,810,4). En los pacientes que recibieron tratamiento antivírico en el plazo de 2 días de la aparición de los síntomas las estancias hospitalarias fueron más breves (media: 4 días frente a 6,5 días; P < 0,05) y la enfermedad tendió a presentar menor gravedad (11,1% frente a 19,0%; P = 0,13). Conclusiones. La investigación activa de los casos hospitalizados permitió una vigilancia casi exhaustiva. Las tasas de hospitalización y la letalidad de la pandemia fueron más moderadas que lo previsto. Durante este brote se confirmó la capacidad de algunas enfermedades subyacentes ya conocidas para aumentar la gravedad. Además, estos resultados demuestran la validez del tratamiento antivírico temprano

First human rabies case in French Guiana, 2008: epidemiological investigation and control.

BACKGROUND: Until 2008, human rabies had never been reported in French Guiana. On 28 May 2008, the French National Reference Center for Rabies (Institut Pasteur, Paris) confirmed the rabies diagnosis, based on hemi-nested polymerase chain reaction on skin biopsy and saliva specimens from a Guianan, who had never travelled overseas and died in Cayenne after presenting clinically typical meningoencephalitis. METHODOLOGY/PRINCIPAL FINDINGS: Molecular typing of the virus identified a Lyssavirus (Rabies virus species), closely related to those circulating in hematophagous bats (mainly Desmodus rotundus) in Latin America. A multidisciplinary Crisis Unit was activated. Its objectives were to implement an epidemiological investigation and a veterinary survey, to provide control measures and establish a communications program. The origin of the contamination was not formally established, but was probably linked to a bat bite based on the virus type isolated. After confirming exposure of 90 persons, they were vaccinated against rabies: 42 from the case's entourage and 48 healthcare workers. To handle that emergence and the local population's increased demand to be vaccinated, a specific communications program was established using several media: television, newspaper, radio. CONCLUSION/SIGNIFICANCE: This episode, occurring in the context of a Department far from continental France, strongly affected the local population, healthcare workers and authorities, and the management team faced intense pressure. This observation confirms that the risk of contracting rabies in French Guiana is real, with consequences for population educational program, control measures, medical diagnosis and post-exposure prophylaxis.

Rapid diagnosis of acute hemorrhagic conjunctivitis due to coxsackievirus A24 variant by real-time one-step RT-PCR.

Coxsackievirus A24 variant is, together with enterovirus 70 and adenoviruses, the major etiological agent involved in acute hemorrhagic conjunctivitis outbreaks worldwide. However, the standard virus isolation method followed by serotyping or VP1 region sequencing is time-consuming. A rapid method for the detection of coxsackievirus A24 variant from conjunctival swab specimens would be useful in the context of explosive and extensive outbreaks. A one-step real-time RT-PCR assay based on TaqMan technology was thus developed and assessed on 36 conjunctival swabs from outbreaks of conjunctivitis in Morocco in 2004 due to a coxsackievirus A24 variant and in Corsica in 2006 due to adenovirus type 3, and 83 virus strains including 41 coxsackievirus A24 variant collected in French Guiana and Guadeloupe in 2003, in the Democratic Republic of the Congo in 2003, in Morocco in 2004 and 42 other virus species genetically close or known to be responsible for conjunctivitis. All the conjunctival swabs from coxsackievirus A24 variant related outbreak and the 41 coxsackievirus A24 variant strains were tested positive by the RT-PCR assay within 4h. This novel single-tube real-time RT-PCR assay is sensitive and specific, and consists in a reliable and faster alternative to the viral culture for recent and future acute hemorrhagic conjunctivitis outbreaks caused by coxsackievirus A24 variant.

Evaluation of an enzyme immunoassay for detection of dengue virus NS1 antigen in human serum.

We evaluated a one-step sandwich-format microplate enzyme immunoassay for detecting dengue virus NS1 antigen (Ag) in human serum by use of Platelia Dengue NS1 Ag kits (Bio-Rad Laboratories, Marnes La Coquette, France). We collected 299 serum samples from patients with dengue disease and 50 serum samples from patients not infected with dengue virus. For the 239 serum samples from patients with acute infections testing positive by reverse transcription-PCR and/or virus isolation for one of the four dengue virus serotypes, the sensitivity of the Platelia Dengue NS1 Ag kit was 88.7% (95% confidence interval, 84.0% to 92.4%). None of the serum samples from patients not infected with dengue virus tested positive with the Platelia Dengue NS1 Ag kit. A diagnostic strategy combining the Platelia Dengue NS1 Ag test for acute-phase sera and immunoglobulin M capture enzyme-linked immunosorbent assay for early-convalescent-phase sera increased sensitivity only from 88.7% to 91.9%. Thus, NS1 antigen detection with the Platelia Dengue NS1 Ag kit could be used for first-line testing for acute dengue virus infection in clinical diagnostic laboratories.

Serological and molecular evidence that human herpesvirus 8 is endemic among Amerindians in French Guiana.

We evaluated the presence of human herpesvirus 8 (HHV-8) infection among groups of Amerindians in French Guiana. The overall prevalence of antibodies against lytic HHV-8 antigens was 23.0% (180/781), increasing from 18.4% in children <6 years old to approximately 30% in older persons (>45 years). Seroprevalence was higher in Amerindians living in remote localities than it was in those living in the coastal region. Analysis of a 725-base pair fragment of the K1 gene amplified from DNA from a Wayampi Amerindian showed that the virus belonged to molecular subtype E, which has hitherto been found in only a few Amerindians in Brazil and Ecuador.