Genetic characterization and pathogenic potential of H10 avian influenza viruses isolated from live poultry markets in Bangladesh.

Fatal human cases of avian-origin H10N8 influenza virus infections have raised concern about their potential for human-to-human transmission. H10 subtype avian influenza viruses (AIVs) have been isolated from wild and domestic aquatic birds across Eurasia and North America. We isolated eight H10 AIVs (four H10N7, two H10N9, one H10N1, and one H10N6) from live poultry markets in Bangladesh. Genetic analyses demonstrated that all eight isolates belong to the Eurasian lineage. HA phylogenetic and antigenic analyses indicated that two antigenically distinct groups of H10 AIVs are circulating in Bangladeshi live poultry markets. We evaluated the virulence of four representative H10 AIV strains in DBA/2J mice and found that they replicated efficiently in mice without prior adaptation. Moreover, H10N6 and H10N1 AIVs caused high mortality with systemic dissemination. These results indicate that H10 AIVs pose a potential threat to human health and the mechanisms of their transmissibility should be elucidated.

Adjuvanting influenza hemagglutinin vaccine with a human pulmonary surfactant-mimicking synthetic compound SF-10 induces local and systemic cell-mediated immunity in mice.

We reported previously that intranasal instillation of a synthetic human pulmonary surfactant with a carboxy vinyl polymer as a viscosity improver, named SF-10, shows potent adjuvanticity for humoral immunity in mice and cynomolgus monkeys. SF-10 effectively induces influenza hemagglutinin vaccine (HAv)-specific IgA in nasal and lung washes and IgG in sera with their neutralizing activities. Since CD8+ T cell-mediated protection is an important requirement for adaptive immunity, we investigated in this study the effects of SF-10 with antigen on local and systemic cell-mediated immunity. Nasal instillation of ovalbumin, a model antigen, combined with SF-10 efficiently delivered antigen to mucosal dendritic and epithelial cells and promoted cross-presentation in antigen presenting cells, yielding a high percentage of ovalbumin-specific cytotoxic T lymphocytes in the nasal mucosa, compared with ovalbumin alone. Nasal immunization of HAv-SF-10 also induced HAv-specific cytotoxic T lymphocytes and upregulated granzyme B expression in splenic CD8+ T cells with their high cytotoxicity against target cells pulsed with HA peptide. Furthermore, nasal vaccination of HAv-SF-10 significantly induced higher cytotoxic T lymphocytes-mediated cytotoxicity in the lungs and cervical lymph nodes in the early phase of influenza virus infection compared with HAv alone. Protective immunity induced by HAv-SF-10 against lethal influenza virus infection was partially and predominantly suppressed after depletion of CD8+ and CD4+ T cells (induced by intraperitoneal injection of the corresponding antibodies), respectively, suggesting that CD4+ T cells predominantly and CD8+ T cells partially contribute to the protective immunity in the advanced stage of influenza virus infection. These results suggest that SF-10 promotes effective antigen delivery to antigen presenting cells, activates CD8+ T cells via cross-presentation, and induces cell-mediated immune responses against antigen.

Immunogenicity and safety of Southern Hemisphere inactivated quadrivalent influenza vaccine: a Phase III, open-label study of adults in Brazil

Abstract The World Health Organization influenza forecast now includes an influenza B strain from each of the influenza B lineages (B/Yamagata and B/Victoria) for inclusion in seasonal influenza vaccines. Traditional trivalent influenza vaccines include an influenza B strain from one lineage, but because two influenza B lineages frequently co-circulate, the effectiveness of trivalent vaccines may be reduced in seasons of influenza B vaccine-mismatch. Thus, quadrivalent vaccines may potentially reduce the burden of influenza compared with trivalent vaccines.In this Phase III, open-label study, we assessed the immunogenicity and safety of Southern Hemisphere inactivated quadrivalent influenza vaccine (Fluarix™ Tetra) in Brazilian adults (NCT02369341). The primary objective was to assess hemagglutination-inhibition antibody responses against each vaccine strain 21 days after vaccination in adults (aged ≥18–60 years) and older adults (aged >60 years). Solicited adverse events for four days post-vaccination, and unsolicited adverse events and serious adverse events for 21 days post-vaccination were also assessed.A total of 63 adults and 57 older adults received one dose of inactivated quadrivalent influenza vaccine at the beginning of the 2015 Southern Hemisphere influenza season. After vaccination, in adults and older adults, the hemagglutination-inhibition titers fulfilled the European licensure criteria for immunogenicity. In adults, the seroprotection rates with HI titer ≥1:40 were 100% (A/H1N1), 98.4% (A/H3N2), 100% (B/Yamagata), and 100% (B/Victoria); in older adults were 94.7% (A/H1N1), 96.5% (A/H3N2), 100% (B/Yamagata), and 100% (B/Victoria). Pain was the most common solicited local adverse events in adults (27/62) and in older adults (13/57), and the most common solicited general adverse events in adults was myalgia (9/62), and in older adults were myalgia and arthralgia (both 2/57). Unsolicited adverse events were reported by 11/63 adults and 10/57 older adults.The study showed that inactivated quadrivalent influenza vaccine was immunogenic and well-tolerated in Brazilian adults and older adults.

Immunogenicity and safety of Southern Hemisphere inactivated quadrivalent influenza vaccine: a Phase III, open-label study of adults in Brazil.

The World Health Organization influenza forecast now includes an influenza B strain from each of the influenza B lineages (B/Yamagata and B/Victoria) for inclusion in seasonal influenza vaccines. Traditional trivalent influenza vaccines include an influenza B strain from one lineage, but because two influenza B lineages frequently co-circulate, the effectiveness of trivalent vaccines may be reduced in seasons of influenza B vaccine-mismatch. Thus, quadrivalent vaccines may potentially reduce the burden of influenza compared with trivalent vaccines. In this Phase III, open-label study, we assessed the immunogenicity and safety of Southern Hemisphere inactivated quadrivalent influenza vaccine (Fluarix™ Tetra) in Brazilian adults (NCT02369341). The primary objective was to assess hemagglutination-inhibition antibody responses against each vaccine strain 21 days after vaccination in adults (aged ≥18-60 years) and older adults (aged >60 years). Solicited adverse events for four days post-vaccination, and unsolicited adverse events and serious adverse events for 21 days post-vaccination were also assessed. A total of 63 adults and 57 older adults received one dose of inactivated quadrivalent influenza vaccine at the beginning of the 2015 Southern Hemisphere influenza season. After vaccination, in adults and older adults, the hemagglutination-inhibition titers fulfilled the European licensure criteria for immunogenicity. In adults, the seroprotection rates with HI titer ≥1:40 were 100% (A/H1N1), 98.4% (A/H3N2), 100% (B/Yamagata), and 100% (B/Victoria); in older adults were 94.7% (A/H1N1), 96.5% (A/H3N2), 100% (B/Yamagata), and 100% (B/Victoria). Pain was the most common solicited local adverse events in adults (27/62) and in older adults (13/57), and the most common solicited general adverse events in adults was myalgia (9/62), and in older adults were myalgia and arthralgia (both 2/57). Unsolicited adverse events were reported by 11/63 adults and 10/57 older adults. The study showed that inactivated quadrivalent influenza vaccine was immunogenic and well-tolerated in Brazilian adults and older adults.

Relationship between haemagglutination inhibition titre and immunity to influenza in ferrets.

Our understanding of the antigenic evolution of the human influenza virus is chiefly derived from experiments in which serum from influenza infected ferrets is tested against panels of virus isolates in the haemagglutination inhibition (HI) assay. The interpretation of these results has been much aided by the development of antigenic mapping techniques, which suppose that the antigenic distance between two different influenza viruses is directly proportional to their fold-difference in titre in this assay. Yet, antigenic distance is not necessarily the same as cross-protection, and high levels of protection have been observed in humans against strains to which they have low HI titres. However, no study has previously addressed the relationship between HI titre and cross-protection in ferrets: the standard animal model. This study fills this gap by analysing published data where pre-challenge HI titres are available for individual ferrets, and post-challenge outcomes have been recorded. Ultimately, this work confirms that it is the absolute, rather than relative, HI titre that determines the extent of immunity and that there is a threshold HI titre beyond which ferrets are completely protected from infection. Nevertheless, this titre is much higher in ferrets than has been suggested for humans. Further, we are consequently able to show that using distance between strains within an antigenic map to predict cross-protection between influenza viruses can be misleading.

Effect of Varying Doses of a Monovalent H7N9 Influenza Vaccine With and Without AS03 and MF59 Adjuvants on Immune Response: A Randomized Clinical Trial.

IMPORTANCE: Human infections with the avian influenza A(H7N9) virus were first reported in China in 2013 and continue to occur. Hemagglutinin H7 administered alone is a poor immunogen necessitating evaluation of adjuvanted H7N9 vaccines. OBJECTIVE: To evaluate the immunogenicity and safety of an inactivated H7N9 vaccine with and without AS03 adjuvant, as well as mixed vaccine schedules that included sequential administration of AS03- and MF59-containing formulations and of adjuvanted and unadjuvanted formulations. DESIGN, SETTING, AND PARTICIPANTS: Double-blind, phase 2 trial at 5 US sites enrolled 980 adults aged 19 through 64 years from September 2013 through November 2013; safety follow-up was completed in January 2015. INTERVENTIONS: The H7N9 vaccine was given on days 0 and 21 at nominal doses of 3.75 µg, 7.5 µg, 15 µg, and 45 µg of hemagglutinin with or without AS03 or MF59 adjuvant mixed on site. MAIN OUTCOMES AND MEASURES: Proportions achieving a hemagglutination inhibition antibody (HIA) titer of 40 or higher at 21 days after the second vaccination; vaccine-related serious adverse events through 12 months after the first vaccination; and solicited signs and symptoms after vaccination through day 7. RESULTS: Two doses of vaccine were required to induce detectable antibody titers in most participants. After 2 doses of an H7N9 formulation containing 15 µg of hemagglutinin given without adjuvant, with AS03 adjuvant, or with MF59 adjuvant, the proportion achieving an HIA titer of 40 or higher was 2% (95% CI, 0%-7%) without adjuvant (n = 94), 84% (95% CI, 76%-91%) with AS03 adjuvant (n = 96), and 57% (95% CI, 47%-68%) with MF59 adjuvant (n = 92) (P < .001 for comparison of the AS03 and MF59 schedules). The 2 schedules alternating AS03-and MF59-adjuvanted formulations led to lower geometric mean titers (GMTs) of (41.5 [95% CI, 31.7-54.4]; n = 92) and (58.6 [95% CI, 44.3-77.6]; n = 96) than the group induced by 2 AS03-adjuvanted formulations (n = 96) (103.4 [95% CI, 78.7-135.9]; P < .001) but higher GMTs than 2 doses of MF59-adjuvanted formulation (n = 94) (29.0 [95% CI, 22.4-37.6]; P < .001). CONCLUSIONS AND RELEVANCE: The AS03 and MF59 adjuvants augmented the immune responses to 2 doses of an inactivated H7N9 influenza vaccine, with AS03-adjuvanted formulations inducing the highest titers. This study of 2 adjuvants used in influenza vaccine formulations with adjuvant mixed on site provides immunogenicity information that may be informative to influenza pandemic preparedness programs. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01942265.

Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

Human antibody responses to avian influenza A(H7N9) virus, 2013.

Understanding host antibody response is crucial for predicting disease severity and for vaccine development. We investigated antibody responses against influenza A(H7N9) virus in 48 serum samples from 21 patients, including paired samples from 15 patients. IgG against subtype H7 and neutralizing antibodies (NAbs) were not detected in acute-phase samples, but ELISA geometric mean titers increased in convalescent-phase samples; NAb titers were 20-80 (geometric mean titer 40). Avidity to IgG against subtype H7 was significantly lower than that against H1 and H3. IgG against H3 was boosted after infection with influenza A(H7N9) virus, and its level in acute-phase samples correlated with that against H7 in convalescent-phase samples. A correlation was also found between hemagglutinin inhibition and NAb titers and between hemagglutinin inhibition and IgG titers against H7. Because of the relatively weak protective antibody response to influenza A(H7N9), multiple vaccinations might be needed to achieve protective immunity.

Serum hemagglutination inhibition activity correlates with protection from gastroenteritis in persons infected with Norwalk virus.

A hemagglutination inhibition (HAI) assay to assess serum antibody responses following Norwalk virus (NV) infection was developed. HAI activity increased significantly in individuals experimentally infected with NV (n = 18) and correlated with antibody levels measured in a histo-blood group antigen (HBGA) blocking assay. Prechallenge HAI antibody levels also correlated with protection from the development of gastroenteritis (Mann-Whitney test, P = 0.02). The HAI assay is another assay suitable for the detection of antibody that correlates with protection from Norwalk virus-associated disease.

[Pandemic influenza 2009 in Russia. Characteristics of the isolation and biological properties of viruses].

Research Institute of Influenza, Ministry of Health and Social Development of Russia, Saint Petersburg The characteristics of the isolation of pandemic influenza A(H1N1)v viruses were studied on chick embryos (CE) and MDCK cell culture. The materials (nasal swabs and autopsies) were collected in different regions Russia in the period from 20 July to 30 December 2009. The paper gives the data of the antigenic analysis of isolates, their capacity to multiply in different species-specific and tissue cell cultures. The viruses isolated on CE were shown to have higher hemagglutination titers and to be more stable. Isolation from the autopsies was effective only on CE. All the test cell lines other than MDCK were insensitive to the isolated pandemic influenza strains. The antigenic analysis showed no significant antigenic drift of the viruses isolated during the first wave of the pandemic in the Russian Federation.

[Flu vaccine and auto-immune and/or inflammatory diseases]. / Vaccination antigrippale chez les patients atteints de maladies systémiques inflammatoires et/ou auto-immunes.

Patients with systemic inflammatory and/or autoimmune diseases have an increased risk of infections particularly severe influenza infections. Annually vaccination can prevent these infections. Available data about the influenza vaccine in these patients show that, it remains well tolerated and effective even if the antibody response is lower compared to healthy controls. These data encourage to vaccine every year patients with systemic inflammatory and/or autoimmune diseases with influenza vaccine, particularly patients taking immunosuppressant drugs or having respiratory, cardiac or renal chronic diseases according to guidelines. More data are needed about the severity of influenza infection and the efficacy of influenza vaccination in patients with systemic inflammatory and/or autoimmune diseases to improve their vaccine coverage.

Protective immunity afforded by inactivated H5N1 (NIBRG-14) vaccine requires antibodies against both hemagglutinin and neuraminidase in mice.

BACKGROUND: Hemagglutination-inhibition (HI) antibody titers correlate with protective immunity to seasonal influenza viruses. However, inactivated H5N1 influenza vaccines from Vietnam 2004 strains afford protection without producing high or even detectable HI antibodies. METHODS: BALB/c mice were immunized twice (at a 3-week interval) with inactivated whole-virus influenza vaccine produced using reverse genetics, with the internal genes of A/PR/8/34 (a high-yield strain) and the hemagglutinin (HA) and neuraminidase (NA) genes of A/Vietnam/1194/04 (H5N1) virus (NIBRG-14) adjuvanted with alum (5 microg of HA). Either HA- or NA-binding antibodies were absorbed from the immune serum. The protective efficacy of these antibodies was determined by injecting the absorbed serum into severe combined immunodeficiency mice, which were then challenged with highly pathogenic H5N1 virus (A/Vietnam/Jp1203/2004; Japanese isolate of A/Vietnam/1203/2004). RESULTS: The NIBRG-14 vaccine elicited levels of anti-HA antibodies similar to levels elicited by the H1N1 vaccines, as well as levels of anti-NA antibodies higher than those elicited by the H1N1 vaccines. The absorption of either anti-HA or anti-NA antibody from immune serum samples obtained from NIBRG-14-vaccinated mice significantly reduced the protective efficacy of the serum. CONCLUSIONS: For NIBRG-14 vaccines to confer protection to vaccinated hosts, both anti-HA and anti-NA antibodies are required. This finding implies that the measurement of both antibody levels may be required for accurate evaluation of vaccine efficacy.

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans.

DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing alpha-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capable of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.

Seroprevalence of egg drop syndrome--76 virus as cause of poor egg productivity of poultry in Nsukka, South East Nigeria.

To determine if egg drop syndrome 76 virus infection is among the causes of lowered egg productivity in commercial poultry farms in South Eastern Part of Nigeria and to know the prevalence of the infection, ten farms with history of lowered egg production in Nsukka local government area of Enugu State were randomly selected. Sera from ten hens in each of the selected farms were assayed for antibodies against EDS 76 virus by the haemagglutination-inhibition (HI) test. The mean HI titre of the ten hens in each of the farms was recorded as EDS - 76 antibody titre for the farm. Nine out of the 10 farms tested were positive for EDS - 76 antibodies with HI titres ranging between 16 and 256. Out of 10 flocks with production of 65% and above 9 were EDS-76 HI negative.

Safety and immunogenicity of an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine: phase I randomised trial.

BACKGROUND: Pathogenic avian influenza A virus H5N1 has caused outbreaks in poultry and migratory birds in Asia, Africa, and Europe, and caused disease and death in people. Although person-to-person spread of current H5N1 strains is unlikely, the virus is a potential source of a future influenza pandemic. Our aim was to assess the safety and immunogenicity of a vaccine against the H5N1 strain. METHODS: We did a randomised, open-label, non-controlled phase I trial in 300 volunteers aged 18-40 years and assigned one of six inactivated split influenza A/Vietnam/1194/2004 (H5N1) influenza vaccine formulations, comprising 7.5 microg (with adjuvant n=50, without adjuvant n=49), 15 microg (n=50, n=50), or 30 microg (n=51, n=50) of haemagglutinin with or without aluminium hydroxide adjuvant. Individuals received two vaccinations (on days 0 and 21) and provided blood samples (on days 0, 21, and 42) for analysis by haemagglutination inhibition and microneutralisation. We recorded all adverse events. Analyses were descriptive. FINDINGS: All formulations were well tolerated, with no serious adverse events, few severe reactions, and no oral temperatures of more than 38 degrees C. All formulations induced an immune response, and responses were detectable in some individuals after only one dose. The adjuvanted 30 microg formulation induced the greatest response (67% haemagglutinin-inhibition seroconversion rate after two vaccinations). Adjuvant did not improve the response to the lower doses. Two vaccinations of non-adjuvanted 7.5 microg, adjuvanted 15 microg, or non-adjuvanted 15 microg seroconverted more than 40% of participants (haemagglutinin-inhibition test only). Haemagglutinin inhibition and neutralising results were comparable. INTERPRETATION: A two-dose regimen with an adjuvanted 30 microg inactivated H5N1 vaccine was safe and showed an immune response consistent with European regulatory requirements for licensure of seasonal influenza vaccine. We noted encouraging responses with lower doses of antigen that need further study to ascertain their relevance for the choice of the final pandemic vaccine.

High level of cross-reactivity in influenza virus hemagglutinin-specific CD4+ T-cell response: implications for the initiation of autoimmune response in multiple sclerosis.

Viral infections play a role in shaping and maintaining the peripheral T-cell repertoire, as well as in the initiation of autoimmune response via mechanisms of molecular mimicry. In this study, we addressed the flexibility of T-cell receptor (TCR) recognition and the degree of structural and sequence homology required for cross-reactive immune response in the induction of autoimmune response. We studied the extent of cross-reactivity of a CD4+T-cell clone (TCC) specific for the immunodominant influenza virus hemagglutinin (Flu-HA) peptide derived from a patient with multiple sclerosis (MS) using positional scanning synthetic peptide combinatorial libraries (PS-SCL). We documented cross-reactivity against 14 Flu-HA variants, 11 viral, 15 human, and 3 myelin-derived peptides. Moreover, we identified six naturally occurring peptides with higher stimulatory potency than the native ligand, implicating high potential for cross-reactivity even for a virus-specific memory TCC. Our study demonstrates that flexibility of TCR recognition is present even in a clone with a high degree of TCR specificity for an infectious agent. The results have implications for vaccine design and for antigen-specific treatment strategies for autoimmune diseases.

Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin.

Haemagglutination-inhibition tests (HI) are used to detect increases in influenza antibody in serum. However, they are relatively insensitive for the detection of human antibody responses to avian haemagglutinin, even in the presence of high titres of neutralising antibody after confirmed infection or vaccination. Human influenza viruses bind preferentially sialic acid containing N-acetylneuraminic acid alpha2,6-galactose (SAalpha2,6Gal) linkages while avian and equine viruses bind preferentially those containing N-acetylneuraminic acid alpha2,3-galactose (SAalpha2,3Gal) linkages. Increasing the proportion of SAalpha2,3Gal linkages on the erythrocytes used, by enzymatic modification or change of species, improves the ability of erythrocytes to bind to avian influenza strains and thereby improves the sensitivity of detection of antibody to avian and equine HA in a range of mammalian and human sera using HI tests.

A passive haemagglutination test for rabies antibodies using rabies glycoprotein coupled erythrocytes.

The presently recommended tests for assaying rabies antibodies like mouse neutralization test (MINT) and rapid fluorescent focus inhibition test (RFFIT) are either time consuming or expensive and are generally performed in reference laboratories. There is a need to develop a specific and rapid method for detection of rabies antibodies that can be used to monitor sero-conversion after pre-or post-exposure vaccination. In this study, we have developed a passive haemagglutination (PHA) using purified rabies virus glycoprotein coupled to sheep erythrocytes using chromium chloride (0.04%) as a coupling agent. Two hundred and fifty five serum samples from people vaccinated with different rabies vaccines, 16 paired serum and CSF samples from autopsy confirmed cases of paralytic rabies, and serum samples from 65 normal healthy controls were tested and evaluated in comparison to standard MNT. Among the vaccinees, 250 samples were positive both by MNT and PHA but 5 samples were negative by PHA and positive by MNT. The titres obtained by PHA were lower compared to MNT, but there was significant correlation between the two (r=0.885). The specificity of the test was 99.7% and sensitivity was 100% as compared to MNT. Thus this PHA test promises to be a rapid and specific test for assaying rabies antibodies and may be useful in screening large number of serum samples for sero conversion after vaccination. It may also assist in rapid laboratory confirmation of paralytic rabies cases, based on detection of antibodies in CSF and serum.

Vaccination of puppies born to immune dams with a canine adenovirus-based vaccine protects against a canine distemper virus challenge.

None of the currently available distemper vaccines provides a satisfactory solution for the immunization of very young carnivores in the face of maternal-derived immunity. Since mucosal immunization with replication-competent adenovirus-based vaccines has been proven effective in the face of passive immunity against the vector, it has the potential to provide a solution for the vaccination of young puppies born to canine distemper virus (CDV)-immune dams. We report the engineering and the characterization of two replication-competent canine adenovirus type 2 (CAV2)-based vaccines expressing, respectively, the CDV hemagglutinin (HA) and fusion (F) antigens. We first demonstrated that the intranasal vaccination with a mixture of both recombinant CAV2s provides an excellent level of protection in seronegative puppies, confirming the value of replication-competent adenovirus-based vectors for mucosal vaccination. In contrast, intranasal immunization with the same vaccine of puppies born to CDV- and CAV2-immune dams, failed to activate specific and protective immune responses. We hypothesized that an active CAV2 infection occurred while puppies were in close contact with the vaccinated dams in the breeding units and that the resulting active mucosal immunity interfered with the intranasal administration of CAV2-based CDV vaccine. However, when puppies born to CDV- and CAV2-immune dams were vaccinated subcutaneously with the CAV2-based CDV vaccine, significant seroconversion and solid protective immunity were triggered despite pre-existing systemic immunity to the vector. This latter result is surprising and suggests that subcutaneous vaccination with a replication-competent recombinant CAV2 may be an efficient strategy to overcome both passive and active adenovirus specific immunity in the dog. From a practical point of view, this could pave the way for an original strategy to vaccinate young puppies in the face of maternal-derived immunity.

Infection of human airway epithelia with H1N1, H2N2, and H3N2 influenza A virus strains.

Three subtypes of influenza A virus cause human disease: H1N1, H2N2, and H3N2. Although all result in respiratory illness, little is known about how these subtypes infect differentiated airway epithelia. Therefore, we assayed A/PR/8/34 (H1N1), A/Japan/305/57 (H2N2), and X31 (H3N2) influenza virus strains for binding and infection on fully differentiated primary cultures of airway epithelia isolated from human bronchus, grown on semiporous filters at an air-liquid interface. In this model system, viral infectivity was highest when virus was applied to the apical versus the basolateral surface; Japan was most infectious, followed by PR8. The X31 strain showed very low levels of infectivity. Confocal microscopy and fluorescence-resonance energy transfer studies indicated that Japan virus could enter and fuse with cellular membranes, while infection with X31 virions was greatly inhibited. Japan virus could also productively infect human trachea explant tissues. These data show that influenza viruses with SAalpha2,3Gal binding specificity, like Japan, productively infect differentiated human airway epithelia from the apical surface. These data are important to consider in the development of pseudotyped recombinant viral vectors for gene transfer to human airway epithelia for gene therapy.