Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Sequential Transmission of Influenza Viruses in Ferrets Does Not Enhance Infectivity and Does Not Predict Transmissibility in Humans.

Airborne transmission in ferrets is a key component of pandemic risk assessment. However, some emerging avian influenza viruses transmit between ferrets but do not spread in humans. Therefore, we evaluated sequential rounds of airborne transmission as an approach to enhance the predictive accuracy of the ferret model. We reasoned that infection of ferrets via the respiratory route and onward transmission would more closely model transmission in humans. We hypothesized that pandemic and seasonal viruses would transmit efficiently over two rounds of transmission, while emerging avian viruses would fail to transmit in a second round. The 2009 pandemic H1N1 (pdm09) and seasonal H3N2 viruses were compared to avian-origin H7N9 and H3N8 viruses. Depending on the virus strain, transmission efficiency varied from 50 to 100% during the first round of transmission; the efficiency for each virus did not change during the second round, and viral replication kinetics in both rounds of transmission were similar. Both the H1N1pdm09 and H7N9 viruses acquired specific mutations during sequential transmission, while the H3N2 and H3N8 viruses did not; however, a global analysis of host-adaptive mutations revealed that minimal changes were associated with transmission of H1N1 and H3N2 viruses, while a greater number of changes occurred in the avian H3N8 and H7N9 viruses. Thus, influenza viruses that transmit in ferrets maintain their transmission efficiency through serial rounds of transmission. This answers the question of whether ferrets can propagate viruses through more than one round of airborne transmission and emphasizes that transmission in ferrets is necessary but not sufficient to infer transmissibility in humans. IMPORTANCE Airborne transmission in ferrets is used to gauge the pandemic potential of emerging influenza viruses; however, some emerging influenza viruses that transmit between ferrets do not spread between humans. Therefore, we evaluated sequential rounds of airborne transmission in ferrets as a strategy to enhance the predictive accuracy of the ferret model. Human influenza viruses transmitted efficiently (>83%) over two rounds of airborne transmission, demonstrating that, like humans, ferrets infected by the respiratory route can propagate the infection onward through the air. However, emerging avian influenza viruses with associated host-adaptive mutations also transmitted through sequential transmission. Thus, airborne transmission in ferrets is necessary but not sufficient to infer transmissibility in humans, and sequential transmission did not enhance pandemic risk assessment.

Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice.

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.

Protective efficacy of influenza group 2 hemagglutinin stem-fragment immunogen vaccines.

The stem of the influenza A virus hemagglutinin (HA) is highly conserved and represents an attractive target for a universal influenza vaccine. The 18 HA subtypes of influenza A are phylogenetically divided into two groups, and while protection with group 1 HA stem vaccines has been demonstrated in animal models, studies on group 2 stem vaccines are limited. Thus, we engineered group 2 HA stem-immunogen (SI) vaccines targeting the epitope for the broadly neutralizing monoclonal antibody CR9114 and evaluated vaccine efficacy in mice and ferrets. Immunization induced antibodies that bound to recombinant HA protein and viral particles, and competed with CR9114 for binding to the HA stem. Mice vaccinated with H3 and H7-SI were protected from lethal homologous challenge with X-79 (H3N2) or A/Anhui/1/2013 (H7N9), and displayed moderate heterologous protection. In ferrets, H7-SI vaccination did not significantly reduce weight loss or nasal wash titers after robust 107 TCID50 H7N9 virus challenge. Epitope mapping revealed ferrets developed lower titers of antibodies that bound a narrow range of HA stem epitopes compared to mice, and this likely explains the lower efficacy in ferrets. Collectively, these findings indicate that while group 2 SI vaccines show promise, their immunogenicity and efficacy are reduced in larger outbred species, and will have to be enhanced for successful translation to a universal vaccine.

In Vitro Neutralization Is Not Predictive of Prophylactic Efficacy of Broadly Neutralizing Monoclonal Antibodies CR6261 and CR9114 against Lethal H2 Influenza Virus Challenge in Mice.

Influenza viruses of the H1N1, H2N2, and H3N2 subtypes have caused previous pandemics. H2 influenza viruses represent a pandemic threat due to continued circulation in wild birds and limited immunity in the human population. In the event of a pandemic, antiviral agents are the mainstay for treatment, but broadly neutralizing antibodies (bNAbs) may be a viable alternative for short-term prophylaxis or treatment. The hemagglutinin stem binding bNAbs CR6261 and CR9114 have been shown to protect mice from severe disease following challenge with H1N1 and H5N1 and with H1N1, H3N2, and influenza B viruses, respectively. Early studies with CR6261 and CR9114 showed weak in vitro activity against human H2 influenza viruses, but the in vivo efficacy against H2 viruses is unknown. Therefore, we evaluated these antibodies against human- and animal-origin H2 viruses A/Ann Arbor/6/1960 (H2N2) (AA60) and A/swine/MO/4296424/06 (H2N3) (Sw06). In vitro, CR6261 neutralized both H2 viruses, while CR9114 only neutralized Sw06. To evaluate prophylactic efficacy, mice were given CR6261 or CR9114 and intranasally challenged 24 h later with lethal doses of AA60 or Sw06. Both antibodies reduced mortality, weight loss, airway inflammation, and pulmonary viral load. Using engineered bNAb variants, antibody-mediated cell cytotoxicity reporter assays, and Fcγ receptor-deficient (Fcer1g-/-) mice, we show that the in vivo efficacy of CR9114 against AA60 is mediated by Fcγ receptor-dependent mechanisms. Collectively, these findings demonstrate the in vivo efficacy of CR6261 and CR9114 against H2 viruses and emphasize the need for in vivo evaluation of bNAbs.IMPORTANCE bNAbs represent a strategy to prevent or treat infection by a wide range of influenza viruses. The evaluation of these antibodies against H2 viruses is important because H2 viruses caused a pandemic in 1957 and could cross into humans again. We demonstrate that CR6261 and CR9114 are effective against infection with H2 viruses of both human and animal origin in mice, despite the finding that CR9114 did not display in vitro neutralizing activity against the human H2 virus. These findings emphasize the importance of in vivo evaluation and testing of bNAbs.

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.

Evaluation of the Biological Properties and Cross-Reactive Antibody Response to H10 Influenza Viruses in Ferrets.

The recent outbreak of avian origin H10N7 influenza among seals in northern Europe and two fatal human infections with an avian H10N8 virus in China have demonstrated that H10 viruses can spread between mammals and cause severe disease in humans. To gain insight into the potential for H10 viruses to cross the species barrier and to identify a candidate vaccine strain, we evaluated the in vitro and in vivo properties and antibody response in ferrets to 20 diverse H10 viruses. H10 virus infection of ferrets caused variable weight loss, and all 20 viruses replicated throughout the respiratory tract; however, replication in the lungs was highly variable. In glycan-binding assays, the H10 viruses preferentially bound "avian-like" α2,3-linked sialic acids. Importantly, several isolates also displayed strong binding to long-chain "human-like" α2,6-linked sialic acids and exhibited comparable or elevated neuraminidase activity relative to human H1N1, H2N2, and H3N2 viruses. In hemagglutination inhibition assays, 12 antisera cross-reacted with ≥14 of 20 H10 viruses, and 7 viruses induced neutralizing activity against ≥15 of the 20 viruses. By combining data on weight loss, viral replication, and the cross-reactive antibody response, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable virus for vaccine development. Collectively, our findings suggest that H10 viruses may continue to sporadically infect humans and other mammals, underscoring the importance of developing an H10 vaccine for pandemic preparedness.IMPORTANCE Avian origin H10 influenza viruses sporadically infect humans and other mammals; however, little is known about viruses of this subtype. Thus, we characterized the biological properties of 20 H10 viruses in vitro and in ferrets. Infection caused mild to moderate weight loss (5 to 15%), with robust viral replication in the nasal tissues and variable replication in the lung. H10 viruses preferentially bind "avian-like" sialic acids, although several isolates also displayed binding to "human-like" sialic acid receptors. This is consistent with the ability of H10 viruses to cross the species barrier and warrants selection of an H10 vaccine strain. By evaluating the cross-reactive antibody response to the H10 viruses and combining this analysis with viral replication and weight loss findings, we identified A/mallard/Portugal/79906/2009 (H10N7) as a suitable H10 vaccine strain.

In Vivo Imaging of Influenza Virus Infection in Immunized Mice.

Immunization is the cornerstone of seasonal influenza control and represents an important component of pandemic preparedness strategies. Using a bioluminescent reporter virus, we demonstrate the application of noninvasive in vivo imaging system (IVIS) technology to evaluate the preclinical efficacy of candidate vaccines and immunotherapy in a mouse model of influenza. Sequential imaging revealed distinct spatiotemporal kinetics of bioluminescence in groups of mice passively or actively immunized by various strategies that accelerated the clearance of the challenge virus at different rates and by distinct mechanisms. Imaging findings were consistent with conclusions derived from virus titers in the lungs and, notably, were more informative than conventional efficacy endpoints in some cases. Our findings demonstrate the reliability of IVIS as a qualitative approach to support preclinical evaluation of candidate medical countermeasures for influenza in mice.IMPORTANCE Influenza A viruses remain a persistent threat to public health. Vaccination and immunotherapy are effective countermeasures for the control of influenza but must contend with antigenic drift and the risk of resistance to antivirals. Traditional preclinical efficacy studies for novel vaccine and pharmaceutical candidates can be time-consuming and expensive and are inherently limited in scope. In vivo imaging approaches offer the potential to noninvasively track virus replication in real time in animal models. In this study, we demonstrate the utility of bioluminescent imaging for tracking influenza virus replication in the lungs of immunized mice and also identify important factors that may influence the accurate interpretation of imaging results. Our findings support the potential of IVIS approaches to enhance traditional preclinical efficacy evaluation of candidate vaccines and human monoclonal antibodies for the prevention and treatment of influenza.

Correction: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm.

[This corrects the article DOI: 10.1371/journal.ppat.1003971.].

Prophylaxis With a Middle East Respiratory Syndrome Coronavirus (MERS-CoV)-Specific Human Monoclonal Antibody Protects Rabbits From MERS-CoV Infection.

With >1600 documented human infections with Middle East respiratory syndrome coronavirus (MERS-CoV) and a case fatality rate of approximately 36%, medical countermeasures are needed to prevent and limit the disease. We examined the in vivo efficacy of the human monoclonal antibody m336, which has high neutralizing activity against MERS-CoV in vitro. m336 was administered to rabbits intravenously or intranasally before infection with MERS-CoV. Prophylaxis with m336 resulted in a reduction of pulmonary viral RNA titers by 40-9000-fold, compared with an irrelevant control antibody with little to no inflammation or viral antigen detected. This protection in rabbits supports further clinical development of m336.

The soft palate is an important site of adaptation for transmissible influenza viruses.

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

Severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume.

UNLABELLED: Ferrets are a valuable model for influenza virus pathogenesis, virus transmission, and antiviral therapy studies. However, the contributions of the volume of inoculum administered and the ferret's respiratory tract anatomy to disease outcome have not been explored. We noted variations in clinical disease outcomes and the volume of inoculum administered and investigated these differences by administering two influenza viruses (A/California/07/2009 [H1N1 pandemic] and A/Minnesota/11/2010 [H3N2 variant]) to ferrets intranasally at a dose of 10(6) 50% tissue culture infective doses in a range of inoculum volumes (0.2, 0.5, or 1.0 ml) and followed viral replication, clinical disease, and pathology over 6 days. Clinical illness and respiratory tract pathology were the most severe and most consistent when the viruses were administered in a volume of 1.0 ml. Using a modified micro-computed tomography imaging method and examining gross specimens, we found that the right main-stem bronchus was consistently larger in diameter than the left main-stem bronchus, though the latter was longer and straighter. These anatomic features likely influence the distribution of the inoculum in the lower respiratory tract. A 1.0-ml volume of inoculum is optimal for delivery of virus to the lower respiratory tract of ferrets, particularly when evaluation of clinical disease is desired. Furthermore, we highlight important anatomical features of the ferret lung that influence the kinetics of viral replication, clinical disease severity, and lung pathology. IMPORTANCE: Ferrets are a valuable model for influenza virus pathogenesis, virus transmission, and antiviral therapy studies. Clinical disease in ferrets is an important parameter in evaluating the virulence of novel influenza viruses, and findings are extrapolated to virulence in humans. Therefore, it is highly desirable that the data from different laboratories be accurate and reproducible. We have found that, even when the same virus was administered at similar doses, different investigators reported a range of clinical disease outcomes, from asymptomatic infection to severe weight loss, ocular and nasal discharge, sneezing, and lethargy. We found that a wide range of inoculum volumes was used to experimentally infect ferrets, and we sought to determine whether the variations in disease outcome were the result of the volume of inoculum administered. These data highlight some less explored features of the model, methods of experimental infection, and clinical disease outcomes in a research setting.

Influenza a virus assembly intermediates fuse in the cytoplasm.

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

Evaluation of three live attenuated H2 pandemic influenza vaccine candidates in mice and ferrets.

UNLABELLED: H2 influenza viruses have not circulated in humans since 1968, and therefore a significant portion of the population would be susceptible to infection should H2 influenza viruses reemerge. H2 influenza viruses continue to circulate in avian reservoirs worldwide, and these reservoirs are a potential source from which these viruses could emerge. Three reassortant cold-adapted (ca) H2 pandemic influenza vaccine candidates with hemagglutinin (HA) and neuraminidase (NA) genes derived from the wild-type A/Japan/305/1957 (H2N2) (Jap/57), A/mallard/6750/1978 (H2N2) (mal/78), or A/swine/MO/4296424/2006 (H2N3) (sw/06) viruses and the internal protein gene segments from the A/Ann Arbor/6/60 ca virus were generated by plasmid-based reverse genetics (Jap/57 ca, mal/78 ca, and sw/06 ca, respectively). The vaccine candidates exhibited the in vitro phenotypes of temperature sensitivity and cold adaptation and were restricted in replication in the respiratory tract of ferrets. In mice and ferrets, the vaccines elicited neutralizing antibodies and conferred protection against homologous wild-type virus challenge. Of the three candidates, the sw/06 ca vaccine elicited cross-reactive antibodies and provided significant protection against the greatest number of heterologous viruses. These observations suggest that the sw/06 ca vaccine should be further evaluated in a clinical trial as an H2 pandemic influenza vaccine candidate. IMPORTANCE: Influenza pandemics arise when novel influenza viruses are introduced into a population with little prior immunity to the new virus and often result in higher rates of illness and death than annual seasonal influenza epidemics. An influenza H2 subtype virus caused a pandemic in 1957, and H2 viruses circulated in humans till 1968. H2 influenza viruses continue to circulate in birds, and the development of an H2 influenza vaccine candidate is therefore considered a priority in preparing for future pandemics. However, we cannot predict whether a human H2 virus will reemerge or a novel avian H2 virus will emerge. We identified three viruses as suitable candidates for further evaluation as vaccines to protect against H2 influenza viruses and evaluated the immune responses and protection that these three vaccines provided in mice and ferrets.

Receptor specificity does not affect replication or virulence of the 2009 pandemic H1N1 influenza virus in mice and ferrets.

Human influenza viruses predominantly bind α2,6 linked sialic acid (SA) while avian viruses bind α2,3 SA-containing complex glycans. Virulence and tissue tropism of influenza viruses have been ascribed to this binding preference. We generated 2009 pandemic H1N1 (pH1N1) viruses with either predominant α2,3 or α2,6 SA binding and evaluated these viruses in mice and ferrets. The α2,3 pH1N1 virus had similar virulence in mice and replicated to similar titers in the respiratory tract of mice and ferrets as the α2,6 and WT pH1N1 viruses. Immunohistochemical analysis determined that all viruses infected similar cell types in ferret lungs. There is increasing evidence that receptor specificity of influenza viruses is more complex than the binary model of α2,6 and α2,3 SA binding and our data suggest that influenza viruses use a wide range of SA moieties to infect host cells.

An open-label phase I trial of a live attenuated H2N2 influenza virus vaccine in healthy adults.

BACKGROUND: Live attenuated influenza vaccines (LAIV) against a variety of strains of pandemic potential are being developed and tested. We describe the results of an open-label phase I trial of a live attenuated H2N2 virus vaccine. OBJECTIVES: To evaluate the safety, infectivity, and immunogenicity of a live attenuated H2N2 influenza virus vaccine. PARTICIPANTS/METHODS: The A/Ann Arbor/6/60 (H2N2) virus used in this study is the attenuated, cold-adapted, temperature-sensitive strain that provides the genetic backbone of seasonal LAIV (MedImmune). We evaluated the safety, infectivity, and immunogenicity of two doses of 10(7) TCID(50) of this vaccine administered by nasal spray 4 weeks apart to normal healthy seronegative adults. RESULTS: Twenty-one participants received a first dose of the vaccine; 18 participants received a second dose. No serious adverse events occurred during the trial. The most common adverse events after vaccination were headache and musculoskeletal pain. The vaccine was restricted in replication: 24% and 17% had virus detectable by culture or rRT-PCR after the first and second dose, respectively. Antibody responses to the vaccine were also restricted: 24% of participants developed an antibody response as measured by either hemagglutination-inhibition assay (10%), or ELISA for H2 HA-specific serum IgG (24%) or IgA (16%) after either one or two doses. None of the participants had a neutralizing antibody response. Vaccine-specific IgG-secreting cells as measured by enzyme-linked immunospot increased from a mean of 0·5 to 2·0/10(6) peripheral blood mononuclear cells (PBMCs); vaccine-specific IgA-secreting cells increased from 0·1 to 0·5/10(6) PBMCs. CONCLUSIONS: The live attenuated H2N2 1960 AA ca vaccine demonstrated a safety profile consistent with seasonal trivalent LAIV but was restricted in replication and minimally immunogenic in healthy seronegative adults.

An open label Phase I trial of a live attenuated H6N1 influenza virus vaccine in healthy adults.

BACKGROUND: We describe the results of an open label Phase I trial of a live attenuated H6N1 influenza virus vaccine (ClinicalTrials.gov Identifier: NCT00734175). METHODS AND FINDINGS: We evaluated the safety, infectivity, and immunogenicity of two doses of 10(7) TCID(50) of the H6N1 Teal HK 97/AA ca vaccine, a cold-adapted and temperature sensitive live, attenuated influenza vaccine (LAIV) in healthy seronegative adults. Twenty-two participants received the first dose of the vaccine, and 18 received the second dose of vaccine 4 weeks later. The vaccine had a safety profile similar to that of other investigational LAIVs bearing avian hemagglutinin (HA) and neuraminidase (NA) genes. The vaccine was highly restricted in replication: two participants had virus detectable by rRT-PCR beyond day 1 after each dose. Antibody responses to the vaccine were also restricted: 43% of participants developed a serum antibody response as measured by any assay: 5% by hemagglutination-inhibition assay, 5% by microneutralization assay, 29% by ELISA for H6 HA-specific IgG and 24% by ELISA for H6 HA specific IgA after either 1 or 2 doses. Following the second dose, vaccine specific IgG and IgA secreting cells as measured by ELISPOT increased from a mean of 0.6 to 9.2/10(6) PBMCs and from 0.2 to 2.2/10(6) PBMCs, respectively. CONCLUSION: The H6N1 LAIV had a safety profile similar to that of LAIV bearing other HA and NA genes, but was highly restricted in replication in healthy seronegative adults. The H6N1 LAIV was also not as immunogenic as the seasonal LAIV.

Comparison of a live attenuated 2009 H1N1 vaccine with seasonal influenza vaccines against 2009 pandemic H1N1 virus infection in mice and ferrets.

The role of seasonal influenza vaccination in pandemic influenza A H1N1 disease is important to address, because a large segment of the population is vaccinated annually. We administered 1 or 2 doses of pandemic H1N1 vaccine (CA/7 ca), a seasonal trivalent inactivated (s-TIV), or live attenuated influenza vaccine (s-LAIV) to mice and ferrets and subsequently challenged them with a pandemic H1N1 virus. In both species, CA/7 ca was immunogenic and conferred complete protection against challenge. s-TIV did not confer protection in either animal model, and s-LAIV did not confer any protection in ferrets. In mice, 2 doses of s-LAIV led to complete protection in the upper respiratory tract and partial protection in the lungs. Our data indicate that vaccination with the seasonal influenza vaccines did not confer complete protection in the lower respiratory tract in either animal model, whereas the CA/7 ca vaccine conferred complete protection in both animal models.

Seasonal influenza infection and live vaccine prime for a response to the 2009 pandemic H1N1 vaccine.

The robust immune response to a single dose of pandemic 2009 H1N1 vaccine suggests that a large segment of the population has been previously primed. We evaluated the effect of seasonal (s) H1N1 infection, s-trivalent inactivated vaccine (s-TIV), and trivalent s-live attenuated influenza vaccine (s-LAIV) before immunization with a pandemic live attenuated influenza vaccine (p-LAIV) in mice. We compared serum and mucosal antibody and pulmonary CD8 and CD4 responses and the virologic response to challenge with a wild-type 2009 pandemic H1N1 (p-H1N1) virus. Two doses of p-LAIV induced cellular immune and robust ELISA and neutralizing antibody responses that were associated with complete protection from p-H1N1 challenge. A single dose of p-LAIV induced a cellular response and ELISA but not a neutralizing antibody response, and incomplete protection from p-H1N1 virus challenge. Primary infection with s-H1N1 influenza virus followed by a dose of p-LAIV resulted in cross-reactive ELISA antibodies and a robust cellular immune response that was also associated with complete protection from p-H1N1 virus challenge. A lower-magnitude but similar response associated with partial protection was seen in mice that received a dose of s-LAIV followed by p-LAIV. Mice that received a dose of s-TIV followed by p-LAIV did not show any evidence of priming. In summary, prior infection with a seasonal influenza virus or s-LAIV primed mice for a robust response to a single dose of p-LAIV that was associated with protection equivalent to two doses of the matched pandemic vaccine.