Evolution of an Eurasian Avian-like Influenza Virus in Naïve and Vaccinated Pigs

Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies

Fatal Cases of Influenza A(H3N2) in Children: Insights from Whole Genome Sequence Analysis

During the Northern Hemisphere winter of 2003–2004 the emergence of a novel influenza antigenic variant, A/Fujian/411/2002-like(H3N2), was associated with an unusually high number of fatalities in children. Seventeen fatal cases in the UK were laboratory confirmed for Fujian/411-like viruses. To look for phylogenetic patterns and genetic markers that might be associated with increased virulence, sequencing and phylogenetic analysis of the whole genomes of 63 viruses isolated from fatal cases and non fatal “control” cases was undertaken. The analysis revealed the circulation of two main genetic groups, I and II, both of which contained viruses from fatal cases. No associated amino acid substitutions could be linked with an exclusive or higher occurrence in fatal cases. The Fujian/411-like viruses in genetic groups I and II completely displaced other A(H3N2) viruses, but they disappeared after 2004. This study shows that two A(H3N2) virus genotypes circulated exclusively during the winter of 2003–2004 in the UK and caused an unusually high number of deaths in children. Host factors related to immune state and differences in genetic background between patients may also play important roles in determining the outcome of an influenza infection

Polymerase basic protein 1 (PB1) as a molecular determinant of fitness and adaptation in influenza a virus

Tese de doutoramento, Farmácia (Microbiologia), Universidade de Lisboa, Faculdade de Farmácia, 2017The World Health Organization and the National Institute of Allergy and Infectious Diseases reported growth deficits of influenza A(H1N1)pdm09 reverse genetic pandemic vaccine virus seeds. These have compromised the effective and timely distribution of vaccines in the 2009 pandemics and accentuated the need to improve the process of vaccine production. In pre-pandemic A(H5N1) research, seed viruses produced by reverse genetics have also been reported to present growth deficits. These deficits have been attributed to a putative sub-optimal protein interaction. The dynamics of the genetic evolution of influenza A viruses appears to suggest a gene segregation pattern between the Polymerase Basic protein 1 (PB1) and antigenic proteins Hemagglutinin (HA) and Neuraminidase (NA). In the reassortment events that lead to the emergence of the 1957 e 1968 pandemic viruses, the contemporary seasonal viruses acquired PB1 genomic segment together with antigenic glycoproteins originating from avian viruses. A similar pattern was identified in 1947, where a reassortment event between seasonal viruses, involving PB1 and antigenic proteins, has altered the epidemiology of the infection to a near-pandemic geographic dispersion. In both situations, viral fitness appears to have benefitted from acquiring a PB1 genomic segment homologous to antigenic proteins. Also, in retrospective studies on the genomic composition of high yield seasonal vaccine seeds produced by classical reassortment, PB1 is frequently co-incorporated with antigenic proteins HA and NA, further suggesting that the interaction between these proteins could have an impact in viral fitness. In this context, we proposed to address the question of PB1 genomic segment being a molecular determinant of fitness and adaptation in influenza A virus and, particularly, of the functional compatibility between PB1 and antigenic proteins being a driver of the overall viral fitness and putatively exploitable to improve seed virus production. The A(H1N1)pdm09 virus was used a model for this research because it is a product of viral reassortment with an unprecedented genomic composition of segments originating from avian, swine and human seasonal viruses. Additionally, the 2009 pandemic vaccine virus presented severe growth deficits and, since the A(H1N1)pdm09 persists in circulation with a seasonal epidemiologic profile, the demand for high yield A(H1N1)pdm09 vaccine seeds will be continuous and the need to adequate the immunogenic strain to the circulating viruses will be recurrent because of antigenic drifts. The objectives of this research were defined as 1) to evaluate the genetic evolution of PB1 in the zoonotic transmission of swine influenza virus and infer its putative contribution towards viral fitness and adaptation, and 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetics A(H1N1)pdm09 vaccine seed model. The approach followed to accomplish objective 1 was to select a study sample of PB1 nucleotide sequences from swine virus that have infected the human host, to analyze phylogeny and mutation trends and to search for putative markers for viral adaptation on the basis of viral molecular epidemiology, genomic location of the polymorphisms and amino-acid properties. Our major findings were that the evolutionary history of PB1 is traceable in terms of lineage and host origin. Specific genomic markers in PB1 appear to putatively relate to the viral adaptation to mammalian hosts, 336I, 361R, 468K and 584Q, and to the viral adaptation to new genomic backgrounds possibly in the sequence of reassortment events, such as 638D and 618D. Residues 298I, 386K and 517V have been found to putatively relate to an enhanced compatibility between PB1 and HA of the H1 subtype, in the mammalian host. A subsequent in vitro investigation of the phenotypic impact of mutations L298I, R386K and I517V acquired by the A(H1N1)pdm09 during its evolutionary history, was performed by generating an A(H1N1)pdm09 recombinant virus and an A(H1N1)pdm09 reassortant in which the specific mutations have been reverted, by reverse genetics. This approach has resulted in two major findings. Acquiring these mutations has been found to putatively promote conformational changes in PB1 and enhance the span of complementary nucleotides possibly involved in PB1 interaction with HA at the RNA level and, on the other hand, has proven detrimental to viral growth kinetics in vitro. These findings have lead us to suggest that the interaction between genomic segments at the RNA level could be a determinant of co-segregation, concordant with a selective packaging model proposed by other authors, but that the mechanisms that drive this process are probably not dependent on a replicative advantage. Our approach to accomplishing objective 2) to determine if the functional or structural compatibility between PB1 and antigenic proteins is a molecular determinant of the overall virus fitness in the reverse genetic A(H1N1)pdm09 vaccine seed model, was to determine the genetic profile of A(H1N1)pdm09 strains circulating in Portugal during the pandemic period and select a prototype immunogenic strain, to generate reassortant viruses with the genomic composition of A(H1N1)pdm09 seed viruses prototypes bearing PB1 homologous and heterologous to antigenic proteins, and to evaluate viral growth and antigen yield in vitro. A sample of specimens collected from the pandemic period in Portugal were evaluated for genetic and phenotypic features and a strain similar to the consensus was selected as a prototype strain. Vaccine seed prototypes of the selected A(H1N1)pdm09 strain in an A/PuertoRico/08/34 backbone were generated by reverse genetics to present the genomic compositions of the 6:2 classical vaccine seed (PR8:HA,NA A(H1N1)pdm09) and a 5:3 seed prototype in which the PB1 segment from the immunogenic strain is co-incorporated with the antigenic proteins (PR8:HA,NA,PB1 A(H1N1)pdm09). Our major findings were that the presence of PB1 homologous to antigenic protein significantly increased viral replication, hemagglutination capacity and Neuraminidase activity. We have establishing proof of concept that, in the PR8:A(H1N1)pdm09 seed virus model, viral growth and antigen yield can be significantly improved by the inclusion of PB1 from the immunogenic strain when compared to the classical seed virus prototype. We consider that, additionally to the role of PB1 protein in viral replication, PB1 genomic segment may be a molecular determinant of the overall virus fitness and a determinant factor in the molecular epidemiology of the viruses by establishing interactions with other segments at the RNA level and by, apparently, being able to genetically change and adapt to improve these interactions. Further research is necessary to clarify the mechanisms of viral genome packaging, the role of interactions at the RNA level in establishing the co-segregation patterns and the specificities of this interactions at the subtype level. However, it becomes clear that the functional compatibility between PB1 and antigenic proteins is a driver of the overall viral fitness in the A(H1N1)pdm09 and is putatively exploitable to improve seed virus production. We also consider that exploring the concept of the compatibility between gene segments or proteins being a determinant factor in the overall viral fitness, can result in major improvements in the production of reverse genetics seed viruses of different influenza subtypes. Also, being aware of the fact that the genomic composition of influenza viruses can have a major phenotypic impact, and that consequently is a determinant of virulence even though the mechanisms that drive the selective packaging remain unclear, we consider that its inclusion in the risk assessment of influenza strains would be extremely relevant for seasonal and pandemic preparedness

The Epidemiology and Control of Human Influenza in Vietnam

Understanding the epidemiology of human influenza in Viet Nam is important for developing local policies and also for understanding the dynamics of influenza in tropical and subtropical southeast Asia. I have analysed an 18 year time-series of influenza-like-illness (ILI) surveillance data, and assessed the relationship of this time-series with climate variables and with sentinel influenza virus surveillance data. I also conducted a study of influenza A/H1N1 transmission within households. ILI notifications in Viet Nam show a latitudinal gradient, with seasonality in the north but no seasonal pattern observed in low lying areas of central and southern Viet Nam. Seasonality is however observed in the elevated provinces of central Viet Nam, suggesting that the seasonal patterns are driven by climate. Principal component analysis finds that temperature and absolute humidity (AH) are positively correlated and together explain around 59% of total climatic variance, and that there is a strong latitudinal gradient in these variables. Regression tree analysis shows that provinces with strong seasonality of AH have strong ILI seasonality. Although virological surveillance data are limited, increases in ILI notifications are associated with an increase in the proportion of upper respiratory tract swabs that are influenza positive. In a prospective study of H1N1/2009 transmission in a household-based cohort, 11 of 59 household contacts were infected, giving a household secondary infection risk of 18.6% (95%CI 10.7-30.4%), but 5 (45%) did not develop symptoms. Virus genetic sequencing indicated that 10 of the 11 secondary cases (91%) were probably infected within the household rather than from the community. This research provides new insights into the seasonality and climatic determinants of ILI and influenza epidemiology in Viet Nam, and on the transmission of influenza within households. The findings are valuable for national influenza control policies and also add to the current state of knowledge of influenza epidemiology

Influenza virus susceptibility to antiviral drugs : drug susceptibility profiling, whole-genome mutational landscape and selective pressure footprints

Tese de doutoramento, Ciências e Tecnologias da Saúde (Microbiologia), Universidade de Lisboa, Faculdade de Medicina, 2018Antivirals play an important and decisive role in the clinical management of influenza and in the underlying reduction of related morbidity and mortality. The emergence of antiviral resistance, and particularly of transmissible resistance, poses a serious threat to public health as it could render influenza antivirals useless against circulating viruses. This is even more worrying when considering the current paucity of alternative antiviral therapy choices. This PhD research project aimed at disclosing the susceptibility of human influenza viruses circulating in Portugal to nationally approved antivirals, and at improving the knowledge on the evolutionary dynamics underlying the emergence and/or spread of influenza variants resistant or with decreased susceptibility to neuraminidase inhibitor (NAI) drugs. To this end, the project focused on three main areas: antiviral susceptibility testing; whole-genome sequencing; and selective pressure (SP) footprints on human influenza neuraminidase (NA)(NAI target). Antiviral susceptibility testing was performed on human influenza viruses circulating in both community and hospital settings from 2004/2005 to 2012/2013, after establishing a technological platform for comprehensive evaluation of virus susceptibility to M2 protein inhibitors and the NAIs oseltamivir (OS) and zanamivir (ZA) (objective 1). Important findings were made on: the circulation of drug-resistant A(H3N2) (M2 inhibitors) and former seasonal (H1N1) (OS) viruses; the cut-off for potentially clinically relevant sub-populations of drug-resistant virus; a potential novel amino acid substitution conferring slightly decreased susceptibility to ZA (N2 NA) and a novel source for a variant with decreased susceptibility; and, the virus type or subtype specificity of two amino acid substitutions conferring reduced susceptibility to the drug. Overall susceptibility data contributed at a better understanding of the relationship between virus NAI susceptibility phenotype and genotype and of the natural variations in the in vitro NAI susceptibility of circulating viruses over time. The emergence of new drift variants (former seasonal A(H1N1), A(H3N2)), the co-circulation of distinct virus lineages (influenza B) and the increase in OS drug use (A(H1N1)pdm09) were found to potentially play a role in this latter. Influenza viruses exhibiting resistance or decreased susceptibility to OS and/or ZA were further evaluated through whole-genome sequencing to identify and characterize the amino acid substitutions specific of their genome (objective 2). No genetic support was found for the fitter NA H275Y OS resistant former seasonal A(H1N1) viruses, but mutations known to or that based on its structural location or functional impact may play a role in the overall viral fitness, were identified in the genome of single or few viruses resistant or with decreased susceptibility to the drug. Large datasets of full-length NA gene sequences of worldwide circulating viruses were created to estimate the global and site-specific SP acting on influenza NA, particularly on the sites associated with NAI resistance or reduced susceptibility and/or contacting with the drug (objective 3a). Further temporal splitting of NA gene sequences allowed to investigate for the first time the impact of NAI introduction into clinic (1999) and/or its increased use during 2009 A(H1N1) pandemic on the SP acting on NA (objective 3b). Major findings include: the potential role of positive SP (PSP) in the low-level and locally variable spread of NA H275Y OS-resistant A(H1N1)pdm09 viruses that has been observed in the community; a potential risk of spread of a synergistic drug-resistant (H275Y/S247N) or a RI (S247G) variant in A(H1N1)pdm09 subtype and a RI variant (A395E) in B/Victoria lineage (positive diversifying selection); and the potential lack of impact of both NAI introduction into clinic and its increased use during 2009 A(H1N1) pandemic on the global and site-specific SP acting on influenza NA, with the single exception of site 154 of B/YAM-lineage NA (framework active site residue). Overall mapping of site-specific SP across the different NA subtypes or lineages allowed for further identify 7 potential new regions for drug targeting. This project marked the beginning of influenza antiviral susceptibility testing and monitoring activities in Portugal. It not only established the technological capacity and capability required to perform such activities but also generated comprehensive information on the susceptibility of circulating human influenza viruses, essential to contribute to both global and European influenza surveillance on antiviral susceptibility. The project also contributed at finding potential determinants of viral fitness in the genome of influenza virus resistant or with decreased susceptibility to NAIs, based on its location onto the protein structure; and at elucidating the role of PSP in the evolutionary pathways to NAI resistance or reduced susceptibility.Fundação Calouste Gulbenkian, projetos FCG 76676 e SDH49; Administração Central do Sistema de Saúde, I.P. (ACSS), projeto SDH4

Avian influenza A virus transmission and the emergence of drug resistance

2011 Fall.Includes bibliographical references.As avian influenza A viruses (AIV) continue to circulate worldwide both naturally, within the reservoir host of wild waterfowl, and cross species barriers, eventually establishing itself in new host species, it is imperative to study the natural reservoir in respect to virus change and transmissibility. This dissertation will focus on the transmissibility of a mallard virus from mallards to other wild and domestic species as well as elucidate the possible outcomes of oseltamivir contamination in the environment and its effect on influenza A virus infected mallards. Low pathogenicity (LP) AIVs of the H5N2 and H7N3 subtypes were utilized to evaluate the ability of transmission of a mallard derived virus to other species present in a co-habitation (barnyard) scenario. Other species in contact with the mallards were chickens, blackbirds, rats, and pigeons. Viral replication was assessed directly from ducks in the barnyard with assessment of the other animals in the barnyard through sero-conversion. Additional animals of each species were directly inoculated with these two viruses and assessed for viral replication. The H5N2 virus was transmitted to other ducks and chickens in the barnyard through either direct or environmental contamination, but not to rats or blackbirds. The H7N3 virus was transmitted to other ducks, chickens, pigeons, and rats. Chickens and blackbirds directly inoculated with both virus strains shed significant amount of virus and seroconverted, but rats and pigeons (except for one pigeon) failed to shed virus but did develop antiviral antibodies. Knowing that both mallard viruses can directly transmit without adaptation, show the mallard to be a good model to further evaluate the outcome of oseltamivir contamination in the environment and its effect on AIV infected mallards. The environment has been shown to be contaminated with significant amounts of oseltamivir carboxylate (OC) in an area of high drug prescription use. We analyzed the outcomes of AIV in infected mallards when they have access to OC in their drinking water. Two separate LPAIV H5N2 viruses were tested for their ability to mutate under drug pressure. One H5N2 virus did not demonstrate any altered sequence after 7-10 days of drug access and infection. The other H5N2 virus did show mutations in the neuraminidase gene that led to an increase in resistance to oseltamivir caused by a specific mutation at E119V. This resistant virus was further evaluated for its ability to transmit between infected and naïve mallards. While the resistant virus did transmit duck to duck, the mutation at position 119 was not detected after challenge or transmission showing instability of this mutation. This could either be a reversion to wild-type or possibly the low level presence of wild-type present in the resistant strain stock that outcompeted with the mutant strain to succeed in the host. This shows, that in these duck experiments, the E119V mutation is not stable in the absence of drug pressure and unlikely to succeed in the host

Strategies for improving Influenza vaccines: insights from the Influenza A H1N1 and SARS-CoV-2 pandemics

Influensa er et luftveisvirus som kan forårsake alvorlig sykdom og under stadig forandring (drift). Dagens influensavaksiner gir begrenset beskyttelse mot slike driftede influensavirus. Dette fører til at vaksinesammensetningen må revurderes på halvårlig basis, og årlig vaksinasjon anbefales til høyrisikogrupper. Målet med denne avhandlingen var å studere immunresponser etter vaksinasjon og infeksjon med influensavirus og koronaviruset Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), med et overordnet mål om å forbedre fremtidige influensavaksiner. Vi studerte antistoffresponser mot overflate-proteinet neuraminidase (NA) på influensaviruset etter infeksjon og vaksinasjon. Vi ville studere dette proteinets potensiale til å aktivere bredere immunresponser og evne til å forbedre eksisterende influensavaksiner. Vi fant at den pandemiske H1N1-vaksinen med AS03-adjuvans induserte langvarige NA-antistoffresponser og at NA var svært immunogent, selv i lave mengder. Vi fant også at årlig sesongvaksinasjon bidro til å vedlikeholde antistofftiter mot NA, men resulterte i reduserte serokonversjon, målt som en stigning i NA-antistoffer. Våre funn støtter bruken av adjuvanser for å øke NA-immunogenisiteten til influensavaksiner, samtidig som de understreker behovet for en standardisering av NA-komponenten i eksisterende vaksiner. Videre ble humane anti-NA monoklonale antistoffer isolert og karakterisert etter pandemisk H1N1-infeksjon i 2009. De monoklonale antistoffene hadde kraftig NA-hemmende aktivitet in vitro og beskyttet også mot infeksjon med influensa A H1N1 og H5N1 virus in vivo. Dette arbeidet ledet til oppdagelsen av en svært konservert epitop på N1 NA, som en kan dra nytte av i fremtidige NA-baserte vaksineformuleringer. Messenger RNA (mRNA)-vaksiner mot SARS-CoV-2 har vært svært vellykkede under Covid-19-pandemien, og mRNA-vaksiner fremstår nå som en lovende vaksineplattform for neste generasjons influensavaksiner. Vi sammenlignet kinetikken og varigheten av immunresponser etter vaksinasjon og infeksjon hos unge og eldre voksne. Våre funn indikerer at eldre kan ha behov for et annerledes vaksinasjonsregime enn yngre for å oppnå en gunstig beskyttelse. Denne innsikten bidrar til å utvikle nye, fremtidige mRNA-vaksinasjonsanbefalinger til denne høyrisikogruppen.Current seasonal influenza vaccines provide limited protection against antigenically drifted influenza viruses. Consequently, vaccine compositions must be re-evaluated on a biannual basis and annual vaccination is recommended for high-risk groups. The objective of this thesis was to study immune responses after vaccination and infection with influenza virus and severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), with the overarching aim of informing strategies for improving future influenza vaccines. Here, we investigated the antibody response to neuraminidase (NA), the second most abundant surface glycoprotein of the influenza virus, after infection and vaccination to explore its potential as a target for broader protection against influenza. We found that AS03 adjuvanted pandemic H1N1 vaccination induced durable NA antibody responses and that NA was highly immunogenic, even with low doses in the vaccine. We also found that repeated seasonal vaccination led to maintenance of NA inhibition (NAI) titres but also to reduced seroconversion rates. Our findings support inclusion of adjuvants to increase NA immunogenicity of influenza vaccines and highlights the need for standardisation of the NA component of current vaccines. Furthermore, we isolated and characterised human anti-NA monoclonal antibodies (mAbs) after pandemic H1N1 infection in 2009. The mAbs had potent NA inhibition activity in vitro and they also protected against lethal challenge with influenza A H1N1 and H5N1 viruses in vivo. This work led to the discovery of a highly conserved epitope on the N1 NA that can guide rational design of future NA-based vaccines. Messenger RNA (mRNA) vaccines against SARS-CoV-2 have been highly successful during the coronavirus disease 19 (COVID-19) pandemic and are now emerging as a promising vaccine platform for next-generation influenza vaccines. We have broadly investigated the kinetics and durability of immune responses after vaccination and infection in young and elderly. Our findings indicate that different vaccination regimen may be needed for optimal protection in older adults and can inform immunisation regimens for mRNA vaccination in this high-risk group in the future.Doktorgradsavhandlin

Influenza vaccine induced antibody responses in relation to evolution of influenza A viruses

Influenza A viruses (IAVs) cause yearly epidemics and occasionally appearing new pandemics. IAVs are subtyped based on their virion surface glycoproteins haemagglutinin and neuraminidase. Seasonal IA epidemics in humans are currently caused by A(H1N1) and A(H3N2) viruses with varying incidence. The natural reservoir of IAVs is birds, and the virus also infects swine and other mammalian species. Global monitoring of the genetic drift and antigenic characteristics of influenza viruses is needed for vaccine strain selection and pandemic preparedness. Vaccine-induced humoral immunity can be analyzed by measuring serum antibodies. In this study, serum antibody levels in adults were assessed before and after vaccination with different seasonal influenza vaccines and pre-pandemic A(H5N1) vaccine candidate viruses. Traditional haemagglutination inhibition (HI) assay was used to measure antibody levels. In addition, a colorimetric microneutralization test (MNT) was optimized to measure functional neutralizing antibodies. The correlation between the methods was found to be high. One of the most effective ways to prevent an infectious disease is vaccination, if circulating viruses are well-matched with vaccine viruses. The protective efficacy of influenza vaccines varies mainly due to virus evolution. In this study we reported reduced cross-protection against drifted A(H3N2) viruses. The latest influenza pandemic was caused by a novel A(H1N1)pdm09 virus, since pre-existing immunity in the majority of human population was missing. After the pandemic, the A(H1N1)pdm09 viruses have continued to circulate as epidemic strains. In this study, seasonal influenza vaccines were found to induce high levels of cross-reactive antibody responses against different genetic group A(H1N1)pdm09 viruses for several years after the pandemic. Avian influenza viruses usually do not easily transmit to humans. However, A(H5N1) viruses have caused serious disease and considerable mortality, which have led to the development of various avian influenza vaccine candidates. Prepandemic A(H5N1) stockpile vaccine was also purchased in Finland. In this study we demonstrated that two heterologous A(H5N1) vaccinations induced longlasting cross-clade humoral immunity.Influenssarokotteiden aikaansaamat vasta-ainevasteet suhteessa influenssa A -virusten muuntumiseen Tarttuvuutensa ja muuntautumiskykynsä vuoksi influenssa A -virukset aiheuttavat vuosittaisia epidemioita ja ajoittain myös maailmanlaajuisia pandemioita. Influenssa A -virukset luokitellaan useisiin alatyyppeihin viruksen kahta eri pintaproteiinia määrittävien hemagglutiniini- ja neuraminidaasigeenien perusteella. A(H1N1)- ja A(H3N2)-alatyypin virukset aiheuttavat vuosittain epidemioita vaihtelevin valtasuhtein. Influenssa A -viruksia esiintyy erityisesti linnuissa, mutta myös sioissa ja joissain muissa nisäkäslajeissa. Influenssavirusten evoluution maailmanlaajuinen seuranta tukee rokotevirusten valintaa ja pandemioihin varautumista. Rokotteen laukaiseman vasta-ainevälitteisen immuniteetin muodostumista voidaan arvioida mittaamalla virusvasta-aineita. Tässä tutkimuksessa tarkasteltiin eri kausi-influenssarokotteiden sekä prepandeemisten A(H5N1)- lintu-influenssarokotteiden synnyttämiä vasta-ainetasoja aikuisilla. Tutkimusmenetelmänä käytettiin perinteistä hemagglutinaation inhibitio -testiä, jonka rinnalle optimoitiin kolorimetrinen mikroneutralisaatiotesti. Menetelmien välinen korrelaatio oli erittäin hyvä. Rokottaminen suojaa tehokkaimmin influenssaa vastaan silloin, kun kiertävät virukset ovat samankaltaisia ennakoidun rokoteviruksen kanssa. Kausi-influenssarokotteiden suojateho vaihtelee virusten evoluutiosta johtuen kaudesta toiseen. Tässä tutkimuksessa tunnistettiin alentunut immuunivaste muuntuneita A(H3N2)-viruksia kohtaan. Viimeisimmän influenssapandemian aiheutti A(H1N1)pdm09-virus, jota vastaan valtaväestöllä ei ollut aiempaa immuniteettia. Pandemian aiheuttaneen viruksen jälkeläisvirukset jäivät kiertämään väestössä normaalina kausi-influenssana. Tässä tutkimuksessa osoitettiin, että kausi-influenssarokotteet saivat aikaan hyvän immuunisuojan eri jälkeläisviruskantoja vastaan useita vuosia pandemian jälkeen. Lintuinfluenssavirukset tarttuvat ihmiseen yleensä huonosti. Vakavampia taudinkuvia ja huomattavaa kuolleisuutta aiheuttaneet A(H5N1)-tartunnat ovat aiheuttaneet huolta uudesta pandemiasta, mistä syystä on kehitetty erilaisia lintuinfluenssarokotekantoja. Prepandeeminen H5N1-mallirokote varattiin myös Suomeen. Tässä tutkimuksessa todettiin, että kahdella, eri viruskantoja sisältävällä H5N1-rokotteella saatiin aikaan laajakirjoinen vasta-ainevälitteinen immuniteetti

Influenza-specific B cell responses in HLA-DR1 transgenic mice

HLA-DR1 transgenic (DR1 Tg) mice provide a model for evaluating the breadth and specificity of CD4 T cell responses that may develop in humans following influenza infection or vaccination. Recent studies identified a tremendously broad HLA-DR1-restricted CD4 T cell responses in DR1 Tg mice infected intranasally with influenza A/New Caledonia/20/99 (NC). In this study, our goals were to characterize B cell responses after NC infection in DR1 Tg mice and establish the correlation between B cell responses and CD4 T cell responses in this system. Influenza-specific B cell responses following virus administration were analyzed in DR1 Tg mice and in the genetically matched H-2b strain C57BL/10J (B10). Following intranasal (i.n.) NC infection, B cell responses in B10 mice featured strong IgG2b and IgG2c production and were typical of previously described B cell responses to a variety of mouse-adapted influenza strains. In contrast, B cell responses in DR1 Tg mice followed delayed kinetics and were strongly skewed to IgG1 production, suggesting the Th2 polarization of CD4 T cell responses. The different antibody isotype profile in DR1 Tg mice compared to B10 mice was evident in antibody secreting cells (ASCs) frequencies and in circulating Abs levels. Surprisingly, although DR1 Tg mice had lower influenza-specific Abs levels, they exhibited higher neutralizing Abs titers early in the response. B cell responses following intranasal infection of influenza A/Puerto Rico/8/1934 (PR8) or intramuscular vaccination of inactivated NC in DR1 Tg mice were different from the observed IgG1 bias after i.n. NC infection. After i.n. PR8 infection, B cell responses were similar in DR1 Tg mice and B10 mice, characterized by predominant IgM/IgG3 production. Additionally, following intramuscular administration of inactivated NC, B cell responses were skewed towards IgG2c production in both DR1 Tg mice and B10 mice, suggesting the Th1 polarization of CD4 T cell responses. A mechanistic understanding of IgG1/Th2 biased B cell responses and better neutralizing Abs production in DR1 Tg mice following i.n. NC infection may have implications for the optimal control of influenza infection

Pandemic Potential of Reassortant Swine Influenza A Viruses

Influenza A viruses are capable of causing disease in several species, including birds, humans and swine. Host specificity of the viruses is not absolute, and is influenced by a range of factors. Swine play a pivotal role in the interspecies transmission of influenza A viruses, as they are susceptible to infection with both human and avian strains and have been implicated as a “mixing vessel” for the reassortment of influenza A viruses from different species. The reassortment of influenza A viruses of human and avian origin led to human influenza pandemics in 1957 and 1968. The dynamics of swine influenza viruses in North America changed drastically with the introduction of the avian-origin PA and PB2 and human-origin HA, NA, and PB1 gene segments and the creation of the triple reassortant swine virus lineage in 1998. While the previously circulating classical swine H1N1 influenza virus lineage was very stable in the swine population, triple reassortant lineage viruses have supplanted the classical H1N1 lineage and undergone repeated reassortment events, acquiring HA and NA genes from human, swine, and avian influenza viruses, while maintaining triple reassortant internal gene (TRIG) cassette. Viruses of the triple reassortant lineage have been very successful in the swine population, yet the mechanisms underlying their unique characteristics and increased fitness have not been elucidated. Here we address the pandemic potential of triple reassortant swine influenza A viruses, their transmissibility, and their relative fitness compared to classical and double reassortant swine influenza viruses. Several triple reassortant viruses, including one with avian-origin HA and NA, were characterized in the ferret, which is a commonly used model for human influenza infection. The effect of the TRIG cassette on the reassortment potential and temperature sensitivity of swine influenza viruses was determined in cell culture, and the replication and transmission of a classical and a reassortant swine virus were compared in pigs. We found that triple reassortant swine viruses replicated efficiently in the ferret model, although there was some variation in transmission efficiencies. An H2N3 virus with avian-origin HA and NA was transmissible in the ferret model, and this transmissibility could be abolished with a single amino acid change in the HA protein that altered its receptor binding specificity. Avian H2N3 viruses were also capable of replicating in ferrets without adaptation and could acquire transmissibility through a change in the receptor binding specificity of the HA protein. Both double and triple reassortant swine viruses had an advantage over the classical H1N1 swine virus at early timepoints in cell culture. Reassortant viruses also demonstrated less temperature sensitivity than the classical H1N1 swine virus. The triple reassortant H1N1 virus had an increased reassortment potential in cell culture compared to the classical swine H1N1 virus as determined by acquisition of a human HA gene. Triple reassortant swine viruses have an increased ability to establish infection, and an increased potential for reassortment, potentially introducing novel HA genes into a host population. This indicates that triple reassortant swine viruses may have an increased potential to cause human pandemics. In April 2009, a novel H1N1 pandemic virus containing five of the six genes of the TRIG cassette emerged in the human population, emphasizing the importance of reassortant swine influenza A viruses in the generation of human pandemics