Standardized In Vitro Models of Human Adipose Tissue Reveal Metabolic Flexibility in Brown Adipocyte Thermogenesis.

Functional human brown and white adipose tissue (BAT and WAT) are vital for thermoregulation and nutritional homeostasis, while obesity and other stressors lead, respectively, to cold intolerance and metabolic disease. Understanding BAT and WAT physiology and dysfunction necessitates clinical trials complemented by mechanistic experiments at the cellular level. These require standardized in vitro models, currently lacking, that establish references for gene expression and function. We generated and characterized a pair of immortalized, clonal human brown (hBA) and white (hWA) preadipocytes derived from the perirenal and subcutaneous depots, respectively, of a 40-year-old male individual. Cells were immortalized with hTERT and confirmed to be of a mesenchymal, nonhematopoietic lineage based on fluorescence-activated cell sorting and DNA barcoding. Functional assessments showed that the hWA and hBA phenocopied primary adipocytes in terms of adrenergic signaling, lipolysis, and thermogenesis. Compared to hWA, hBA were metabolically distinct, with higher rates of glucose uptake and lactate metabolism, and greater basal, maximal, and nonmitochondrial respiration, providing a mechanistic explanation for the association between obesity and BAT dysfunction. The hBA also responded to the stress of maximal respiration by using both endogenous and exogenous fatty acids. In contrast to certain mouse models, hBA adrenergic thermogenesis was mediated by several mechanisms, not principally via uncoupling protein 1 (UCP1). Transcriptomics via RNA-seq were consistent with the functional studies and established a molecular signature for each cell type before and after differentiation. These standardized cells are anticipated to become a common resource for future physiological, pharmacological, and genetic studies of human adipocytes.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 µg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 µg HA/dose + AS03A; 3. inactivated vaccine with 11.5 µg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 µg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Influenza virus NS1- C/EBPß gene regulatory complex inhibits RIG-I transcription.

Influenza virus non-structural protein 1 (NS1) counteracts host antiviral innate immune responses by inhibiting Retinoic acid inducible gene-I (RIG-I) activation. However, whether NS1 also specifically regulates RIG-I transcription is unknown. Here, we identify a CCAAT/Enhancer Binding Protein beta (C/EBPß) binding site in the RIG-I promoter as a repressor element, and show that NS1 promotes C/EBPß phosphorylation and its recruitment to the RIG-I promoter as a C/EBPß/NS1 complex. C/EBPß overexpression and siRNA knockdown in human lung epithelial cells resulted in suppression and activation of RIG-I expression respectively, implying a negative regulatory role of C/EBPß. Further, C/EBPß phosphorylation, its interaction with NS1 and occupancy at the RIG-I promoter was associated with RIG-I transcriptional inhibition. These findings provide an important insight into the molecular mechanism by which influenza NS1 commandeers RIG-I transcriptional regulation and suppresses host antiviral responses.

Meeting report and review: Immunological assays and correlates of protection for next-generation influenza vaccines.

BACKGROUND: This report summarizes the discussions and conclusions from the "Immunological Assays and Correlates of Protection for Next-Generation Influenza Vaccines" meeting which took place in Siena, Italy, from March 31, 2019, to April 2, 2019. CONCLUSIONS: Furthermore, we review current correlates of protection against influenza virus infection and disease and their usefulness for the development of next generation broadly protective and universal influenza virus vaccines.

Repeated vaccination against matched H3N2 influenza virus gives less protection than single vaccination in ferrets.

Epidemiological studies suggest that humans who receive repeated annual immunization with influenza vaccine are less well protected against influenza than those who receive vaccine in the current season only. To better understand potential mechanisms underlying these observations, we vaccinated influenza-naive ferrets either twice, 10 months apart (repeated vaccination group; RV), or once (current season only group; CS), using a prime-boost regimen, and then challenged the ferrets with A/Hong Kong/4801/2014(H3N2). Ferrets that received either vaccine regimen were protected against influenza disease and infection relative to naive unvaccinated ferrets, but the RV group shed more virus, especially at the peak of virus shedding 2 days post infection (p < 0.001) and regained weight more slowly (p < 0.05) than those in the CS group. Qualitative, rather than quantitative, differences in the antibody response may affect protection after repeated influenza vaccination.

Heterologous prime-boost with A(H5N1) pandemic influenza vaccines induces broader cross-clade antibody responses than homologous prime-boost.

Highly pathogenic avian influenza (HPAI) A(H5Nx) viruses continue to pose a pandemic threat. US national vaccine stockpiles are a cornerstone of the influenza pandemic preparedness plans. However, continual genetic and antigenic divergence of A(H5Nx) viruses requires the development of effective vaccination strategies using stockpiled vaccines and adjuvants for pandemic preparedness. Human sera collected from healthy adults who received either homologous (2 doses of a AS03A-adjuvanted A/turkey/Turkey/1/2005, A/Turkey), or heterologous (primed with AS03A-adjuvanted A/Indonesia/5/2005, A/Indo, followed by A/Turkey boost) prime-boost vaccination regimens were analyzed by hemagglutination inhibition and microneutralization assays against 8 wild-type HPAI A(H5Nx) viruses from 6 genetic clades. Molecular, structural and antigenic features of the A(H5Nx) viruses that could influence the cross-clade antibody responses were also explored. Compared with homologous prime-boost vaccinations, priming with a clade 2.1.3.2 antigen (A/Indo) followed by one booster dose of a clade 2.2.1 antigen (A/Turkey) administered 18 months apart did not compromise the antibody responses to the booster vaccine (A/Turkey), it also broadened the cross-clade antibody responses to several antigenically drifted variants from 6 heterologous clades, including an antigenically distant A(H5N8) virus (A/gyrfalcon/Washington/410886/2014, clade 2.3.4.4) that caused recent outbreaks in US poultry. The magnitude and breadth of the cross-clade antibody responses against emerging HPAI A(H5Nx) viruses are associated with genetic, structural and antigenic differences from the vaccine viruses and enhanced by the inclusion of an adjuvant. Heterologous prime-boost vaccination with AS03A adjuvanted vaccine offers a vaccination strategy to use existing stockpiled vaccines for pandemic preparedness against new emerging HPAI A(H5Nx) viruses.

The Effect of Preexisting Immunity on Virus Detection and Immune Responses in a Phase II, Randomized Trial of a Russian-Backbone, Live, Attenuated Influenza Vaccine in Bangladeshi Children.

BACKGROUND: In a 2012 Phase II clinical trial, 300 Bangladeshi children aged 24 to 59 months with no prior influenza vaccine exposure were randomized to receive a single intranasally-administered dose of either trivalent, Russian-backbone, live, attenuated influenza vaccine (LAIV) or placebo. Protocol-defined analyses, presented in the companion manuscript, demonstrate decreased viral detection and immunogenicity for A/H1N1pdm09, relative to the A/H3N2 and B strains. This post hoc analysis of the trial data aims to investigate the LAIV strain differences by testing the hypothesis that preexisting humoral and mucosal immunity may influence viral recovery and immune responses after LAIV receipt. METHODS: We used logistic regressions to evaluate the relations between markers of preexisting immunity (ie, hemagglutination inhibition [HAI], microneutralization, and immunoglobulin G and immunoglobulin A (both serum and mucosal antibodies) and LAIV viral recovery in the week post-vaccination. We then tested for potential effect modification by baseline HAI titers (ie, <10 versus ≥10) and week 1 viral recovery on the LAIV-induced serum and mucosal immune responses, measured between days 0 and 21 post-vaccination. RESULTS: Higher levels of preexisting immunity to influenza A/H3N2 and B were strongly associated with strain-specific prevention of viral shedding upon LAIV receipt. While evidence of LAIV immunogenicity was observed for all 3 strains, the magnitudes of immune responses were most pronounced in children with no evidence of preexisting HAI and in those with detectable virus. CONCLUSIONS: The results provide evidence for a bidirectional association between viral replication and immunity, and underscore the importance of accounting for preexisting immunity when evaluating virologic and immunologic responses to LAIVs. CLINICAL TRIALS REGISTRATION: NCT01625689.

Immunogenicity and Viral Shedding of Russian-Backbone, Seasonal, Trivalent, Live, Attenuated Influenza Vaccine in a Phase II, Randomized, Placebo-Controlled Trial Among Preschool-Aged Children in Urban Bangladesh.

BACKGROUND: We evaluated a Russian-backbone, live, attenuated influenza vaccine (LAIV) for immunogenicity and viral shedding in a randomized, placebo-controlled trial among Bangladeshi children. METHODS: Healthy children received a single, intranasal dose of LAIV containing the 2011-2012 recommended formulation or placebo. Nasopharyngeal wash (NPW) specimens were collected on days 0, 2, 4, and 7. Reverse transcription polymerase chain reactions and sequencing identified the influenza virus (vaccine or wild-type). On days 0 and 21, blood specimens were collected to assess immunogenicity using hemagglutination inhibition, microneutralization, and immunoglobulin A (IgA) and G enzyme-linked immunosorbent assays (ELISAs); NPW specimens were also collected to assess mucosal immunogenicity using kinetic IgA ELISA. RESULTS: We enrolled 300 children aged 24 through 59 months in the immunogenicity and viral shedding analyses. Among children receiving LAIV, 45% and 67% shed A/H3N2 and B vaccine strains, respectively. No child shed A/H1N1 vaccine strain. There were significantly higher day 21 geometric mean titers (GMTs) for the LAIV, as compared to the placebo groups, in all immunoassays for A/H3N2 and B (log10 titer P < .0001; GMT Ratio >2.0). Among immunoassays for A/H1N1, only the mucosal IgA GMT was significantly higher than placebo at day 21 (log10 titer P = .0465). CONCLUSIONS: Children vaccinated with LAIV had serum and mucosal antibody responses to A/H3N2 and B, but only a mucosal IgA response to A/H1N1. Many children shed A/H3N2 and B vaccine strains, but none shed A/H1N1. More research is needed to determine the reason for decreased LAIV A/H1N1 immunogenicity and virus shedding. CLINICAL TRIALS REGISTRATION: NCT01625689.

Cell culture-derived influenza vaccines in the severe 2017-2018 epidemic season: a step towards improved influenza vaccine effectiveness.

The 2017-2018 seasonal influenza epidemics were severe in the US and Australia where the A(H3N2) subtype viruses predominated. Although circulating A(H3N2) viruses did not differ antigenically from that recommended by the WHO for vaccine production, overall interim vaccine effectiveness estimates were below historic averages (33%) for A(H3N2) viruses. The majority (US) or all (Australian) vaccine doses contained multiple amino-acid changes in the hemagglutinin protein, resulting from the necessary adaptation of the virus to embryonated hen's eggs used for most vaccine manufacturing. Previous reports have suggested a potential negative impact of egg-driven substitutions on vaccine performance. With BARDA support, two vaccines licensed in the US are produced in cell culture: recombinant influenza vaccine (RIV, Flublok™) manufactured in insect cells and inactivated mammalian cell-grown vaccine (ccIIV, Flucelvax™). Quadrivalent ccIIV (ccIIV4) vaccine for the 2017-2018 influenza season was produced using an A(H3N2) seed virus propagated exclusively in cell culture and therefore lacking egg adaptative changes. Sufficient ccIIV doses were distributed (but not RIV doses) to enable preliminary estimates of its higher effectiveness relative to the traditional egg-based vaccines, with study details pending. The increased availability of comparative product-specific vaccine effectiveness estimates for cell-based and egg-based vaccines may provide critical clues to inform vaccine product improvements moving forward.

Influence of Immune Priming and Egg Adaptation in the Vaccine on Antibody Responses to Circulating A(H1N1)pdm09 Viruses After Influenza Vaccination in Adults.

Background: Although ferret antisera used in influenza surveillance did not detect antigenic drift of A(H1N1)pdm09 viruses during the 2015-2016 season, low vaccine effectiveness was reported in adults. We investigated the immune basis of low responses to circulating A(H1N1)pdm09 viruses after vaccination. Methods: Prevaccination and postvaccination serum samples collected from >300 adults (aged 18-49 years) in 6 seasons (2010-2011 to 2015-2016) were analyzed using hemagglutination inhibition assays to evaluate the antibody responses to 13 A(H1N1) viruses circulated from 1977 to 2016. Microneutralization and serum adsorption assays were used to verify the 163K and 223R specificity of antibodies. Results: Individual antibody profiles to A(H1N1) viruses revealed 3 priming patterns: USSR/77, TW/86, or NC/99 priming. More than 20% of adults had reduced titers to cell-propagated circulating 6B.1 and 6B.2 A(H1N1)pdm09 viruses compared with the A/California/07/2009 vaccine virus X-179A. Significantly reduced antibody reactivity to circulating viruses bearing K163Q was observed only in the USSR/77-primed cohort, whereas significantly lower reactivity caused by egg-adapted Q223R change was detected across all 3 cohorts. Conclusion: Both 163K specificity driven by immune priming and 223R specificity from egg-adapted changes in the vaccine contributed to low responses to circulating A(H1N1)pdm09 viruses after vaccination. Our study highlights the need to incorporate human serology in influenza surveillance and vaccine strain selection.

Development of a high-throughput assay to detect antibody inhibition of low pH induced conformational changes of influenza virus hemagglutinin.

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.

Human infection with avian influenza A(H7N2) virus-Virginia, 2002.

BACKGROUND: In March 2002, an outbreak of low-pathogenic avian influenza (LPAI) A(H7N2) was detected among commercial poultry operations in Virginia. METHODS: We performed a serosurvey of 80 government workers involved in efforts to control the outbreak. RESULTS: One study participant who assisted with disposal of infected birds tested positive for neutralizing antibodies to influenza A(H7N2) by microneutralization assay and H7-specific IgM antibodies by enzyme-linked immunosorbent assay (ELISA). The acute infection was temporally associated with an influenza-like illness that resolved without hospitalization. CONCLUSION: This study documents the earliest evidence of human infection with an H7 influenza virus of the North American lineage.

Kinetics of Serological Responses in Critically Ill Patients Hospitalized With 2009 Pandemic Influenza A(H1N1) Virus Infection in Canada, 2009-2011.

Background: The kinetics of the antibody response during severe influenza are not well documented. Methods: Critically ill patients infected with 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09), confirmed by reverse-transcription polymerase chain reaction analysis or seroconversion (defined as a ≥4-fold rise in titers), during 2009-2011 in Canada were prospectively studied. Antibody titers in serially collected sera were determined using hemagglutinin inhibition (HAI) and microneutralization assays. Average antibody curves were estimated using linear mixed-effects models and compared by patient outcome, age, and corticosteroid treatment. Results: Of 47 patients with A(H1N1)pdm09 virus infection (median age, 47 years), 59% had baseline HAI titers of <40, and 68% had baseline neutralizing titers of <40. Antibody titers rose quickly after symptom onset, and, by day 14, 83% of patients had HAI titers of ≥40, and 80% had neutralizing titers ≥40. Baseline HAI titers were significantly higher in patients who died compared with patients who survived; however, the antibody kinetics were similar by patient outcome and corticosteroid treatment. Geometric mean titers over time in older patients were lower than those in younger patients. Conclusions: Critically ill patients with influenza A(H1N1)pdm09 virus infection had strong HAI and neutralizing antibody responses during their illness. Antibody kinetics differed by age but were not associated with patient outcome.

Report on eighth WHO meeting on development of influenza vaccines that induce broadly protective and long-lasting immune responses: Chicago, USA, 23-24 August 2016.

In August 2016, the World Health Organization (WHO) convened the "Eighth meeting on development of influenza vaccines that induce broadly protective and long-lasting immune responses" to discuss the regulatory requirements and pathways for licensure of next-generation influenza vaccines, and to identify areas where WHO can promote the development of such vaccines. Participants included approximately 120 representatives of academia, the vaccine industry, research and development funders, and regulatory and public health agencies. They reviewed the draft WHO preferred product characteristics (PPCs) of vaccines that could address prioritized unmet public health needs and discussed the challenges facing the development of such vaccines, especially for low- and middle-income countries (LMIC). They defined the data desired by public-health decision makers globally and explored how to support the progression of promising candidates into late-stage clinical trials and for all countries. This report highlights the major discussions of the meeting.

Update: Increase in Human Infections with Novel Asian Lineage Avian Influenza A(H7N9) Viruses During the Fifth Epidemic - China, October 1, 2016-August 7, 2017.

Among all influenza viruses assessed using CDC's Influenza Risk Assessment Tool (IRAT), the Asian lineage avian influenza A(H7N9) virus (Asian H7N9), first reported in China in March 2013,* is ranked as the influenza virus with the highest potential pandemic risk (1). During October 1, 2016-August 7, 2017, the National Health and Family Planning Commission of China; CDC, Taiwan; the Hong Kong Centre for Health Protection; and the Macao CDC reported 759 human infections with Asian H7N9 viruses, including 281 deaths, to the World Health Organization (WHO), making this the largest of the five epidemics of Asian H7N9 infections that have occurred since 2013 (Figure 1). This report summarizes new viral and epidemiologic features identified during the fifth epidemic of Asian H7N9 in China and summarizes ongoing measures to enhance pandemic preparedness. Infections in humans and poultry were reported from most areas of China, including provinces bordering other countries, indicating extensive, ongoing geographic spread. The risk to the general public is very low and most human infections were, and continue to be, associated with poultry exposure, especially at live bird markets in mainland China. Throughout the first four epidemics of Asian H7N9 infections, only low pathogenic avian influenza (LPAI) viruses were detected among human, poultry, and environmental specimens and samples. During the fifth epidemic, mutations were detected among some Asian H7N9 viruses, identifying the emergence of high pathogenic avian influenza (HPAI) viruses as well as viruses with reduced susceptibility to influenza antiviral medications recommended for treatment. Furthermore, the fifth-epidemic viruses diverged genetically into two separate lineages (Pearl River Delta lineage and Yangtze River Delta lineage), with Yangtze River Delta lineage viruses emerging as antigenically different compared with those from earlier epidemics. Because of its pandemic potential, candidate vaccine viruses (CVV) were produced in 2013 that have been used to make vaccines against Asian H7N9 viruses circulating at that time. CDC is working with partners to enhance surveillance for Asian H7N9 viruses in humans and poultry, to improve laboratory capability to detect and characterize H7N9 viruses, and to develop, test and distribute new CVV that could be used for vaccine production if a vaccine is needed.

Cross-Reactive Antibody Responses to Novel H5Nx Influenza Viruses Following Homologous and Heterologous Prime-Boost Vaccination with a Prepandemic Stockpiled A(H5N1) Vaccine in Humans.

Recently, novel highly pathogenic avian influenza H5Nx viruses (clade 2.3.4.4) caused outbreaks in US poultry. We evaluated the potential of a stockpiled A(H5N1) A/Anhui/1/2005 (clade 2.3.4) vaccine to elicit cross-reactive antibody responses to these emerging viruses. Sera from subjects who received 2 doses of MF59-adjuvanted A/Anhui/1/2005, or 1 dose of MF59-adjuvanted A/Anhui/1/2005 following priming with a clade 1 vaccine were characterized by microneutralization assays and modified hemagglutination inhibition (HI) assays. Only heterologous prime-boost vaccination induced modest cross-reactive HI antibody responses to H5Nx viruses. Heterologous prime-boost may provide a more effective vaccination strategy to broaden the antibody responses to emerging viruses.

Effect of Priming With Seasonal Influenza A(H3N2) Virus on the Prevalence of Cross-Reactive Hemagglutination-Inhibition Antibodies to Swine-Origin A(H3N2) Variants.

Background: Recent outbreaks of swine-origin influenza A(H3N2) variant (H3N2v) viruses have raised public health concerns. Previous studies indicated that older children and young adults had the highest levels of hemagglutination-inhibition (HI) antibodies to 2010-2011 H3N2v viruses. However, newly emerging 2013 H3N2v have acquired antigenic mutations in the hemagglutinin at amino acid position 145 (N145K/R). We estimated the levels of serologic cross-reactivity among humans primed with seasonal influenza A(H3N2) (sH3N2), using postinfection ferret antisera. We also explored age-related HI antibody responses to 2012-2013 H3N2v viruses. Methods: Human and ferret antisera were tested in HI assays against 1 representative 2012 H3N2v (145N) and 2 2013 H3N2v (145K/R) viruses, together with 9 sH3N2 viruses circulating since 1968. Results: Low levels of cross-reactivity between the H3N2v and sH3N2 viruses from the 1970s-1990s were observed using postinfection ferret antisera. The overall seroprevalence among the sH3N2-primed population against 2012-2013 H3N2v viruses was >50%, and age-related seroprevalence was observed. Seroprevalence was significantly higher to 2013 H3N2v than to 2012 H3N2v viruses among some children likely to have been primed with A/Sydney/5/97-like (145K) or A/Wuhan/359/95-like viruses (145K). Conclusions: A single substitution (N145K/R) was sufficient to affect seropositivity to H3N2v viruses in some individuals. Insight into age-related antibody responses to newly emerging H3N2v viruses is critical for risk assessment and pandemic preparedness.

Pathogenicity testing of influenza candidate vaccine viruses in the ferret model.

The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

Infection with influenza A(H1N1)pdm09 during the first wave of the 2009 pandemic: Evidence from a longitudinal seroepidemiologic study in Dhaka, Bangladesh.

BACKGROUND: We determined influenza A(H1N1)pdm09 antibody levels before and after the first wave of the pandemic in an urban community in Dhaka, Bangladesh. METHODS: We identified a cohort of households by stratified random sampling. We collected baseline serum specimens during July-August 2009, just prior to the initial wave of the 2009 pandemic in this community and a second specimen during November 2009, after the pandemic peak. Paired sera were tested for antibodies against A(H1N1)pdm09 virus using microneutralization assay and hemagglutinin inhibition (HI) assay. A fourfold increase in antibody titer by either assay with a titer of ≥40 in the convalescent sera was considered a seroconversion. At baseline, an HI titer of ≥40 was considered seropositive. We collected information on clinical illness from weekly home visits. RESULTS: We tested 779 paired sera from the participants. At baseline, before the pandemic wave, 1% overall and 3% of persons >60 years old were seropositive. After the first wave of the pandemic, 211 (27%) individuals seroconverted against A(H1N1)pdm09. Children aged 5-17 years had the highest proportion (37%) of seroconversion. Among 264 (34%) persons with information on clinical illness, 191 (72%) had illness >3 weeks prior to collection of the follow-up sera and 73 (38%) seroconverted. Sixteen (22%) of these 73 seroconverted participants reported no clinical illness. CONCLUSION: After the first pandemic wave in Dhaka, one in four persons were infected by A(H1N1)pdm09 virus and the highest burden of infection was among the school-aged children. Seroprevalence studies supplement traditional surveillance systems to estimate infection burden.