HIV Drug Resistance in Kenyan Infants Diagnosed With HIV and Their Mothers.

The presence and type of HIV drug resistance mutations among 5 infants diagnosed with HIV were assessed and compared with their mothers' viral mutations. Mother and infant blood samples were sequenced and screened for HIV drug resistance mutations using the Stanford HIV Sequence Database. Three of 5 (60%) mother-infant pairs harbored HIV drug resistance mutations.

Maternal viral load monitoring: Coverage and clinical action at 4 Kenyan hospitals.

BACKGROUND: Kenya's guidelines for prevention of mother-to-child transmission of HIV (PMTCT) recommend routine viral load (VL) monitoring for pregnant and breastfeeding women. METHOD: We assessed PMTCT VL monitoring and clinical action occurring between last menstrual period (LMP) and 6 months postpartum at 4 Kenyan government hospitals. Pregnant women enrolled in the HIV Infant Tracking System from May 2016-March 2018 were included. We computed proportions who received VL testing within recommended timeframes and who received clinical action after unsuppressed VL result. RESULTS: Of 424 participants, any VL testing was documented for 305 (72%) women and repeat VL testing was documented for 79 (19%). Only 115 women (27%) received a guideline-adherent baseline VL test and 27 (6%) received a guideline-adherent baseline and repeat VL test sequence. Return of baseline and repeat VL test results to the facility was high (average 96%), but patient notification of VL results was low (36% baseline and 49% repeat). Clinical action for unsuppressed VL results was even lower: 11 of 38 (29%) unsuppressed baseline results and 2 of 14 (14%) unsuppressed repeat results triggered clinical action. DISCUSSION: Guideline-adherent VL testing and clinical intervention during PMTCT must be prioritized to improve maternal care and reduce the risk of HIV transmission to infants.

Using a Social Support Framework to Understand How HIV Positive Kenyan Men Engage in PMTCT/EID Care: Qualitative Insights From Male Partners.

Greater male partner involvement in Prevention of Mother to Child Transmission (PMTCT) and Early Infant Diagnosis (EID) is associated with improved outcomes. Perceived low social support for the mother can negatively impact the uptake of PMTCT/EID services. Most research relies on women's reports of the types and quality of male partner support received versus what is desired. This qualitative study examines Kenyan male partners' reported social support provision pre- and post-partum from their own perspective. The study was embedded within intervention development studies in Kenya designed to develop and pilot a PMTCT module of a web based system to improve EID. Focus groups were conducted with male partners of pregnant women with HIV and elicited feedback on male partner involvement in maternal and child care and factors affecting participation. Interviews were analyzed within a theoretical social support framework. Participants described providing tangible support (financial resources), informational support (appointment reminders) and emotional support (stress alleviation in the face of HIV-related adversity). African conceptualizations of masculinity and gender norms influenced the types of support provided. Challenges included economic hardship; insufficient social support from providers, peers and bosses; and HIV stigma. Collaboration among providers, mothers and partners; a community-based social support system; and recasting notions of traditional masculinity were identified as ways to foster male partner support.

Predictors of Mortality Among HIV-exposed Infants Through 18 Months of Age in Kenya: A Retrospective Review of Programmatic Data.

We identified mortality predictors among HIV-exposed uninfected infants and infants living with HIV in Kenyan early infant diagnosis services between 2012 and 2017. Younger maternal age and absence of antenatal antiretroviral therapy among HIV-exposed uninfected infants (n = 2366) and travel time to hospital and delayed infant testing among infants living with HIV (n = 130) predicted mortality, highlighting the importance of supporting engagement in maternal/pediatric HIV services.

eHealth Interventions for Early Infant Diagnosis: Mothers' Satisfaction with the HIV Infant Tracking System in Kenya.

The HIV Infant Tracking System (HITSystem) is an eHealth intervention to improve early infant diagnosis (EID) through alerts to providers and text messages to mothers. This study explored mothers' experiences receiving standard and HITSystem-enhanced EID services to assess perceived intervention benefits, acceptability, and opportunities for improvement. This qualitative study was embedded within a cluster-randomized control trial to evaluate the HITSystem at six Kenyan government hospitals (3 intervention, 3 control). We conducted semi-structured interviews with 137 mothers attending EID follow-up visits. Compared to control sites, participants at HITSystem sites described enhanced EID quality; HITSystem-generated texts informed them of result availability and retesting needs, provided cues-to-action for clinic attendance, and engendered opportunities for patient support. They described improved EID efficiency through shorter waiting periods for results and fewer hospital visits. Participants reported high satisfaction with EID and acceptability of text messages; however, modifications to ensure text delivery, increase repeat testing reminders, include low literacy content options, and provide encouraging messages were suggested. These user experience data suggest improvements in EID at HITSystem sites when compared with control sites.

Infant HIV testing at birth using point-of-care and conventional HIV DNA PCR: an implementation feasibility pilot study in Kenya.

BACKGROUND: Infant HIV diagnosis by HIV DNA polymerase chain reaction (PCR) testing at the standard 6 weeks of age is often late to mitigate the mortality peak that occurs in HIV positive infants' first 2-3 months of life. Kenya recently revised their early infant diagnosis (EID) guidelines to include HIV DNA PCR testing at birth (pilot only), 6 weeks, 6 months, and 12 months postnatal and a final 18-month antibody test. The World Health Organization (WHO) approved point-of-care (POC) diagnostic platforms for infant HIV testing in resource-limited countries that could simplify logistics and expedite infant diagnosis. Sustainable scale-up and optimal utility in Kenya and other high-prevalence countries depend on robust implementation studies in diverse clinical settings. METHODS: We will pilot the implementation of birth testing by HIV DNA PCR, as well as two POC testing systems (Xpert HIV-1 Qual [Xpert] and Alere q HIV-1/2 Detect [Alere q]), on specimens collected from Kenyan infants at birth (0 to 2 weeks) and 6 weeks (4 to < 24 weeks) postnatal. The formative phase will inform optimal implementation of birth testing and two POC testing technologies. Qualitative interviews with stakeholders (providers, parents of HIV-exposed infants, and community members) will assess attitudes, barriers, and recommendations to optimize implementation at their respective sites. A non-blinded pilot study at four Kenyan hospitals (n = 2 Xpert, n = 2 Alere q platforms) will evaluate infant HIV POC testing compared with standard of care HIV DNA PCR testing in both the birth and 6-week windows. Objectives of the pilot are to assess uptake, efficiency, quality, implementation variables, user experiences of birth testing with both POC testing systems or with HIV DNA PCR, and costs. DISCUSSION: This study will generate data on the clinical impact and feasibility of adding HIV testing at birth utilizing POC and traditional PCR HIV testing strategies in resource-limited settings. Data from this pilot will inform the optimal implementation of Kenya's birth testing guidelines and of POC testing systems for the improvement of EID outcomes. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03435887. Registered 26 February 2018.

"Closing the Gap": Provider Recommendations for Implementing Birth Point of Care HIV Testing.

Delays in traditional HIV DNA PCR testing for early infant diagnosis (EID) at 6 weeks of age result in late antiretroviral treatment (ART). Birth point of care (POC) testing is an emerging strategy with the potential to streamline EID services. We elicited providers' recommendations for introducing birth POC testing to guide strategies in Kenya and similar settings. We conducted formative interviews with 26 EID providers from four Kenyan hospitals prior to POC implementation. Providers discussed the need for comprehensive training, covering both EID and POC-specific topics for all key personnel. Providers highlighted equipment considerations, such as protocols for maintenance and safe storage. Providers emphasized the need for maternal counseling to ensure patient acceptance and most agreed that specimen collection for birth POC testing should occur in the maternity department and supported a multidisciplinary approach. Though most providers supported ART initiation based on a positive birth POC result, a few expressed concerns with result validity. To maximize implementation success, provider training, equipment security, maternal counseling, and logistics of testing must be planned and communicated to providers.

Evaluation of the HIV Infant Tracking System (HITSystem) to optimise quality and efficiency of early infant diagnosis: a cluster-randomised trial in Kenya.

BACKGROUND: The HIV Infant Tracking System (HITSystem) is a web-based intervention linking providers of early infant diagnosis, laboratory technicians, and mothers and infants to improve outcomes for HIV-exposed infants. We aimed to evaluate the efficacy of the HITSystem on key outcomes of early infant diagnosis. METHODS: We did a cluster-randomised trial at six hospitals in Kenya, which were matched on geographic region, resource level, and volume of patients (high, medium, and low). We randomly allocated hospitals within a matched pair to either the HITSystem (intervention; n=3) or standard of care (control; n=3). A random number generator was used to assign clusters. Investigators were unaware of the randomisation process. Eligible participants were mothers aged 18 years or older with an infant younger than 24 weeks presenting for their first early infant diagnosis appointment. The primary outcome was complete early infant diagnosis retention, which was defined as receipt of all indicated age-specific interventions until 18 months post partum (for HIV-negative infants) or antiretroviral therapy initiation (for HIV-positive infants). Analysis was per protocol in all randomised pairs judged eligible, excluding infant deaths and those who moved or were transferred to another health facility. Modified intention-to-treat sensitivity analyses judged all infant deaths and transfers as incomplete early infant diagnosis retention. Separate multivariable logistic regression analyses were done with intervention group, hospital volume, and significant covariates as fixed effects. This trial is registered with ClinicalTrials.gov, number NCT02072603; the trial has been completed. FINDINGS: Between Feb 16, 2014, and Dec 31, 2015, 895 mother-infant pairs were enrolled. Of these, 87 were judged ineligible for analysis, 26 infants died, and 92 pairs moved or were transferred to another health facility. Thus, data from 690 mother-infant pairs were analysed, of whom 392 were allocated to the HITSystem and 298 to standard of care. Mother-infant pairs were followed up to Sept 30, 2017. Infants diagnosed as HIV-positive were followed up for a median of 2·1 months (IQR 1·6-4·8) and HIV-negative infants were followed up for a median of 17·0 months (IQR 16·6-17·6). Infants enrolled in the HITSystem were significantly more likely to receive complete early infant diagnosis services compared with those assigned standard of care (334 of 392 [85%] vs 180 of 298 [60%]; adjusted odds ratio [OR] 3·7, 95% CI 2·5-5·5; p<0·0001). No intervention effect was recorded at high-volume hospitals, but strong effects were seen at medium-volume and low-volume hospitals. Modified intention-to-treat analyses for complete early infant diagnosis were also significant (334 of 474 [70%] vs 180 of 334 [54%]; adjusted OR 2·0, 95% CI 1·4-2·7; p<0·0001). No adverse events related to study participation were reported. INTERPRETATION: The HITSystem intervention is effective and feasible to implement in low-resource settings. The HITSystem algorithms have been modified to include HIV testing at birth, and an adapted HITSystem 2.0 version is supporting HIV-positive pregnant women to prevent perinatal transmission and optimise maternal and infant outcomes. FUNDING: National Institute of Child Health and Human Development.

The type of adjuvant in whole inactivated influenza a virus vaccines impacts vaccine-associated enhanced respiratory disease.

Influenza A virus (IAV) causes a disease burden in the swine industry in the US and is a challenge to prevent due to substantial genetic and antigenic diversity of IAV that circulate in pig populations. Whole inactivated virus (WIV) vaccines formulated with oil-in-water (OW) adjuvant are commonly used in swine. However, WIV-OW are associated with vaccine-associated enhanced respiratory disease (VAERD) when the hemagglutinin and neuraminidase of the vaccine strain are mismatched with the challenge virus. Here, we assessed if different types of adjuvant in WIV vaccine formulations impacted VAERD outcome. WIV vaccines with a swine δ1-H1N2 were formulated with different commercial adjuvants: OW1, OW2, nano-emulsion squalene-based (NE) and gel polymer (GP). Pigs were vaccinated twice by the intramuscular route, 3 weeks apart, then challenged with an H1N1pdm09 three weeks post-boost and necropsied at 5 days post infection. All WIV vaccines elicited antibodies detected using the hemagglutination inhibition (HI) assay against the homologous vaccine virus, but not against the heterologous challenge virus; in contrast, all vaccinated groups had cross-reactive IgG antibody and IFN-γ responses against H1N1pdm09, with a higher magnitude observed in OW groups. Both OW groups demonstrated robust homologous HI titers and cross-reactivity against heterologous H1 viruses in the same genetic lineage. However, both OW groups had severe immunopathology consistent with VAERD after challenge when compared to NE, GP, and non-vaccinated challenge controls. None of the WIV formulations protected pigs from heterologous virus replication in the lungs or nasal cavity. Thus, although the type of adjuvant in the WIV formulation played a significant role in the magnitude of immune response to homologous and antigenically similar H1, none tested here increased the breadth of protection against the antigenically-distinct challenge virus, and some impacted immunopathology after challenge.

Factors affecting induction of peripheral IFN-γ recall response to influenza A virus vaccination in pigs.

While T cell contribution to IAV immunity is appreciated, data comparing methods to evaluate IFN-γ production by IAV-specific T cells elicited following vaccination is limited. To understand the differential immunogenicity between live-attenuated influenza virus (LAIV) and whole-inactivated virus (WIV) vaccines in relation to induction of peripheral T cell responses, ELISpot and ELISA were used to assess IFN-γ production by peripheral lymphocytes following antigen restimulation. Following restimulation, peripheral blood lymphocytes from WIV-vaccinated pigs had a greater quantity of IFN-γ secreting cells (SC) and IFN-γ secreted compared to LAIV vaccinated and non-vaccinated (NV) pigs. Pig age at time of WIV vaccination significantly impacted peripheral IAV-specific IFN-γ recall response, as did the inclusion of adjuvant in the WIV vaccine. Collectively, these data indicate that peripheral IAV-specific IFN-γ recall responses are not predictive of LAIV vaccination status, thus, are unlikely to be a useful surrogate for evaluating LAIV immunogenicity and predicting cross-protection. However, these data suggest that the evaluation of peripheral IFN-γ recall responses may be useful for identifying factors, such as animal age or vaccine formulation, that may impact parenterally-delivered WIV vaccine immunogenicity. Overall, results did not differ based upon the assay used to evaluate IFN-γ recall responses. Therefore, either ELISpot or ELISA could serve as a measure for evaluating IAV-specific IFN-γ cell-mediated immune responses in swine.

Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type.

The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and respiratory specimens of pigs vaccinated with intramuscular whole-inactivated virus (WIV), intranasal live-attenuated influenza virus (LAIV), and intranasal wild type (WT) IAV. NI titers were also analyzed in sera from an investigation of piglet vaccination in the presence of passive maternally-derived antibodies. Test antigens contained genetically divergent swine-lineage NA genes homologous or heterologous to the vaccines with mismatched hemagglutinin genes (HA). Naïve piglets responded to WIV and LAIV vaccines and WT infection with strong homologous serum NI titers. Cross-reactivity to heterologous NAs depended on the degree of genetic divergence between the NA genes. Bronchoalveolar lavage specimens of LAIV and WT-immunized groups also had significant NI titers against the homologous antigen whereas the WIV group did not. Piglets of vaccinated sows received high levels of passive NI antibody, but their NI responses to homologous LAIV vaccination were impeded. These data demonstrate the utility of the enzyme-linked lectin assay for efficient NI antibody titration of serum as well as respiratory tract secretions. Swine IAV vaccines that induce robust NI responses are likely to provide broader protection against the diverse and rapidly evolving IAV strains that circulate in pig populations. Mucosal antibodies to NA may be one of the protective immune mechanisms induced by LAIV vaccines.

Vaccine-associated enhanced respiratory disease is influenced by haemagglutinin and neuraminidase in whole inactivated influenza virus vaccines.

Multiple subtypes and many antigenic variants of influenza A virus (IAV) co-circulate in swine in the USA, complicating effective use of commercial vaccines to control disease and transmission. Whole inactivated virus (WIV) vaccines may provide partial protection against IAV with substantial antigenic drift, but have been shown to induce vaccine-associated enhanced respiratory disease (VAERD) when challenged with an antigenic variant of the same haemagglutinin (HA) subtype. This study investigated the role the immune response against HA, neuraminidase (NA) and nucleoprotein (NP) may play in VAERD by reverse engineering vaccine and challenge viruses on a common backbone and using them in a series of vaccination/challenge trials. Mismatched HA between vaccine and challenge virus was necessary to induce VAERD. However, vaccines containing a matched NA abrogated the VAERD phenomenon induced by the HA mismatch and this was correlated with NA-inhibiting (NI) antibodies. Divergence between the two circulating swine N2 lineages (92 % identity) resulted in a loss of NI cross-reactivity and also resulted in VAERD with the mismatched HA. The NP lineage selected for use in the WIV vaccine strains did not affect protection or pathology. Thus the combination of HA and NA in the vaccine virus strains played a substantial role in vaccine protection versus immunopathology, suggesting that vaccines that target the HA protein alone could be more prone to VAERD due to the absence of cross-protective NI antibodies.

Heterologous challenge in the presence of maternally-derived antibodies results in vaccine-associated enhanced respiratory disease in weaned piglets.

Control of influenza A virus (IAV) in pigs is done by vaccination of females to provide maternally-derived antibodies (MDA) through colostrum. Our aim was to evaluate if MDA interfere with IAV infection, clinical disease, and transmission in non-vaccinated piglets. In the first study, naïve sows were vaccinated with H1N2-δ1 whole inactivated virus (WIV) vaccine. In a follow-up study seropositive sows to 2009 pandemic H1N1 (H1N1pdm09) were boosted with H1N1pdm09 WIV or secondary experimental infection (EXP). MDA-positive pigs were challenged with homologous or heterologous virus, and MDA-negative control groups were included. WIV-MDA piglets were protected from homologous infection. However, piglets with WIV-derived MDA subsequently challenged with heterologous virus developed vaccine associated enhanced respiratory disease (VAERD), regardless of history of natural exposure in the sows. Our data indicates that although high titers of vaccine-derived MDA reduced homologous virus infection, transmission, and disease, MDA alone was sufficient to induce VAERD upon heterologous infection.

Optimal Use of Vaccines for Control of Influenza A Virus in Swine.

Influenza A virus in swine (IAV-S) is one of the most important infectious disease agents of swine in North America. In addition to the economic burden of IAV-S to the swine industry, the zoonotic potential of IAV-S sometimes leads to serious public health concerns. Adjuvanted, inactivated vaccines have been licensed in the United States for over 20 years, and there is also widespread usage of autogenous/custom IAV-S vaccines. Vaccination induces neutralizing antibodies and protection against infection with very similar strains. However, IAV-S strains are so diverse and prone to mutation that these vaccines often have disappointing efficacy in the field. This scientific review was developed to help veterinarians and others to identify the best available IAV-S vaccine for a particular infected herd. We describe key principles of IAV-S structure and replication, protective immunity, currently available vaccines, and vaccine technologies that show promise for the future. We discuss strategies to optimize the use of available IAV-S vaccines, based on information gathered from modern diagnostics and surveillance programs. Improvements in IAV-S immunization strategies, in both the short term and long term, will benefit swine health and productivity and potentially reduce risks to public health.

An optimized enzyme-linked lectin assay to measure influenza A virus neuraminidase inhibition antibody titers in human sera.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. The traditional thiobarbituric acid (TBA) method to quantify NA inhibiting antibodies is cumbersome and not suitable for routine serology. An enzyme-linked lectin assay (ELLA) described by Lambre et al. (1990) is a practical alternative method for measuring NA inhibition (NI) titers. This report describes optimization of the ELLA for measuring NI titers in human sera against influenza A viruses, using H6N1 and H6N2 viruses as antigens. The optimized ELLA is subtype-specific and reproducible. While the titers measured by ELLA are somewhat greater than those measured by a miniaturized TBA method, seroconversion rates are the same, suggesting similarity in assay sensitivity under these optimized conditions. The ELLA described in this report provides a practical format for routine evaluation of human antibody responses to NA.

Divergent immune responses and disease outcomes in piglets immunized with inactivated and attenuated H3N2 swine influenza vaccines in the presence of maternally-derived antibodies.

Live-attenuated influenza virus (LAIV) prime-boost vaccination previously conferred protection against heterologous H3N2 swine influenza challenge, including in piglets with maternally derived antibodies (MDA). Conversely, a whole-inactivated virus (WIV) vaccine was associated with enhanced disease. This study was aimed at identifying immune correlates of cross-protection. Piglets with and without MDA received intramuscular adjuvanted WIV or intranasal LAIV, and were challenged with heterologous H3N2. WIV induced cross-reactive IgG, inhibited by MDA, and a moderate T cell response. LAIV elicited mucosal antibodies and T cells cross-reactive to the heterologous challenge strain. The presence of MDA at LAIV vaccination blocked lung and nasal antibody production, but did not interfere with T cell priming. Even without mucosal antibodies, MDA-positive LAIV vaccinates were protected, indicating a likely role for T cells. Based on the data, one LAIV dose can induce cell-mediated immunity against antigenically divergent H3N2 influenza virus despite passive antibody interference with humoral immune responses.

Analyzing swine sera for functional antibody titers against influenza A neuraminidase proteins using an enzyme-linked lectin assay (ELLA).

Neuraminidase (NA) is an envelope glycoprotein of influenza viruses, including swine-lineage influenza A viruses. NA possesses sialidase activity, which is functionally important at multiple points in viral replication, counter-balancing the sialic acid receptor binding activity of the hemagglutinin (HA), the other major envelope glycoprotein. The NA proteins of influenza A viruses have been classified into nine serological subtypes, and they undergo antigenic drift variation similar to that of HA. Antibodies to NA are analyzed much less often than antibodies to HA. The conventional assay for NA inhibition (NI) antibody titration, established decades ago, is widely considered unwieldy and inefficient for routine use. In recent years, a few new formats have been developed which still measure inhibition of NA enzymatic function, but more efficiently and with less chemical waste produced. Described here is the enzyme-linked lectin assay (ELLA), which is performed in 96-well plates and analyzed on a spectrophotometric plate reader. An important factor in adoption of the ELLA technique for animal studies, such as swine, is the choice of NA antigen, which may be purified protein or whole virus containing an antigenically irrelevant HA protein. This NI assay, in conjunction with the hemagglutination inhibiting (HI) antibody assay, offers a practical way to characterize viral isolates more fully and to quantify antibodies induced by infection or vaccination.

Live attenuated influenza vaccine provides superior protection from heterologous infection in pigs with maternal antibodies without inducing vaccine-associated enhanced respiratory disease.

Control of swine influenza A virus (IAV) in the United States is hindered because inactivated vaccines do not provide robust cross-protection against the multiple antigenic variants cocirculating in the field. Vaccine efficacy can be limited further for vaccines administered to young pigs that possess maternally derived immunity. We previously demonstrated that a recombinant A/sw/Texas/4199-2/1998 (TX98) (H3N2) virus expressing a truncated NS1 protein is attenuated in swine and has potential for use as an intranasal live attenuated influenza virus (LAIV) vaccine. In the present study, we compared 1 dose of intranasal LAIV with 2 intramuscular doses of TX98 whole inactivated virus (WIV) with adjuvant in weanling pigs with and without TX98-specific maternally derived antibodies (MDA). Pigs were subsequently challenged with wild-type homologous TX98 H3N2 virus or with an antigenic variant, A/sw/Colorado/23619/1999 (CO99) (H3N2). In the absence of MDA, both vaccines protected against homologous TX98 and heterologous CO99 shedding, although the LAIV elicited lower hemagglutination inhibition (HI) antibody titers in serum. The efficacy of both vaccines was reduced by the presence of MDA; however, WIV vaccination of MDA-positive pigs led to dramatically enhanced pneumonia following heterologous challenge, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). A single dose of LAIV administered to MDA-positive pigs still provided partial protection from CO99 and may be a safer vaccine for young pigs under field conditions, where dams are routinely vaccinated and diverse IAV strains are in circulation. These results have implications not only for pigs but also for other influenza virus host species.

Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines.

Vaccines are instrumental in controlling the burden of influenza virus infection in humans and animals. Antibodies raised against both major viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), can contribute to protective immunity. Vaccine-induced HA antibodies have been characterized extensively, and they generally confer protection by blocking the attachment and fusion of a homologous virus onto host cells. Although not as well characterized, some functions of NA antibodies in influenza vaccine-mediated immunity have been recognized for many years. In this review, we summarize the case for NA antibodies in influenza vaccine-mediated immunity. In the absence of well-matched HA antibodies, NA antibodies can provide varying degrees of protection against disease. NA proteins of seasonal influenza vaccines have been shown in some instances to elicit serum antibodies with cross-reactivity to avian-origin and swine-origin influenza strains, in addition to HA drift variants. NA-mediated immunity has been linked to (i) conserved NA epitopes amongst otherwise antigenically distinct strains, partly attributable to the segmented influenza viral genome; (ii) inhibition of NA enzymatic activity; and (iii) the NA content in vaccine formulations. There is a potential to enhance the effectiveness of existing and future influenza vaccines by focusing greater attention on the antigenic characteristics and potency of the NA protein.

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs.

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4(+)CD8(-), CD4(+)CD8(+), CD4(-)CD8(+), and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4(+)CD8(+) cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains.