Serum neutralizing antibody and lymphocyte transformation responses after influenza B virus infections

Serum neutralizing antibody and influenza B-specific lymphocyte blast transformation responses were measured in 110 adults and children after an influenza B outbreak. Serum neutralizing antibody and lymphocyte blast transformation responses were seen in 67 to 75% of adults and children recently infected (less than 1 year), but significant lymphocyte blast transformation responses were seen in only 25% of those whose infection was remote (greater than or equal to 3 years). The frequencies of influenza B-induced lymphocyte blast transformation and serum neutralizing antibody responses were similar in the adults and children with similar infection histories.     

Comparison of complement fixation and hemagglutination inhibition assays for detecting antibody responses following influenza virus vaccination

Complement fixation (CF) was compared to hemagglutination inhibition (HI) as a method for identifying antibody responses to influenza virus vaccination. CF assays were performed at two different laboratories using paired (pre- and postvaccination) sera from 38 vaccinated laboratory employees; HI assays were performed at a third laboratory. As expected, most vaccinees (31/38 = 82%) responded to at least one of three influenza virus antigens as measured by HI. In contrast, only 21% (8/38) of vaccinees showed a response by CF at laboratory 1, and only 29% (11/38) showed a response by CF at laboratory 2. These findings indicate that due to low sensitivity, CF assays should not be used to assess the antibody response to influenza virus vaccination.     

Comparison of hemagglutination inhibition assay, an ELISA-based micro-neutralization assay and colorimetric microneutralization assay to detect antibody responses to vaccination against influenza A H1N1 2009 virus

The hemagglutination inhibition (HI) assay has been the main method used to investigate immune responses to vaccination against influenza H1N1 (2009) virus. However microneutralization tests (MNT) have been shown to be more sensitive and more specific. In this study, the three methods of choice: (i) the HI assay, (ii) an ELISA-based conventional MNT and (iii) a colorimetric MNT in terms of their ability to detect antibody responses in serum pairs collected from 43 healthy individuals before and 21 days after vaccination were compared. The colorimetric MNT was established yielding intra- and inter-run imprecisions of 7.5% and 12.4%, respectively. Testing of antisera to seasonal influenza viruses demonstrated the assay to be specific for antibodies to influenza H1N1 (2009) virus. A good correlation between the three methods was found, being highest for the ELISA-MNT and the colorimetric MNT (r = 0.714 for geometric mean titers (GMT) and r = 0.695 for titer increases). Similar rates of fourfold titer increases were detected: 95.3% in the ELISA-MNT vs. 93.0% in colorimetric MNT and 95.3% in HI assay. The ELISA-based MNT demonstrated the highest titer range leading to the highest postvaccination GMT and the highest titer increase (>50-fold). The lowest GMTs were measured with the HI assay, while the colormetric MNT detected the highest GMT in prevaccination sera. Taken together, similar seroconversion rates were obtained with the three assays. The ELISA-MNT appeared to be the best method to compare absolute pre- and postvaccination GMTs. The colorimetric MNT, being less labour-intensive than the ELISA-MNT, seems to be a suitable tool in vaccination studies.     

Lymphocyte blastogenic responses to influenza virus antigens after influenza infection and vaccination in humans.

Virus-specific in vitro cell-mediated immune responses were investigated in 20 normal volunteers who were challenged with liver influenza A/VIC/3/75 (H3N2) virus and in 13 volunteers who were vaccinated with inactivated vaccine containing A/VIC and A/NJ/8/76 (HswN1) antigens. Lymphocyte cultures were established from peripheral blood samples obtained prior to and at various times after infection or vaccination. Blastogenesis was determined by [3H]thymidine incorporation after stimulation of cultures with purified, inactivated, whole influenza viruses. Six days after infection, significantly elevated levels of blastogenesis were observed after in vitro stimulation with A viruses of hemagglutinin and neuraminidase subtypes that were the same as (H3N2) or antigenically distinct from (Heq1Neq1 or HswN1) those of the challenge virus, although maximum stimulation was noted with virus of the same hemagglutinin subtype (H3) as the challenge virus. Similar although more prolonged blastogenic responses were noted in lymphocyte cultures from vaccinees who had serum antibody rises after vaccination. The kinetics of these responses suggest that cell-mediated immunity may play a role in early events after infection and vaccination with influenza virus.     

Differential neutralizing antibody responses to varicella-zoster virus glycoproteins B and E following naked DNA immunization.

The only available vaccine against varicella-zoster virus (VZV) consists of the VZV-Oka attenuated but persistent virus strain. Development of a safer, subunit vaccine is therefore desirable. In this prospect, nucleic acid vaccines, expressing truncated forms of VZV glycoproteins B (recgB) and E (recgE) from which the anchor and the cytoplasmic domains were deleted, were used to immunize mice. Vaccination with recgB encoding plasmid elicited a strong and specific humoral immune response. Total IgG and neutralizing titres were comparable to those previously obtained by vaccination with purified and adjuvanted native recgB. In contrast, mice immunization with recgE encoding plasmid only induced a very weak immune response whereas we previously showed that vaccination with adjuvanted native or denatured recgE protein led to high neutralizing titres. The weakness of the immune response induced by recgE-encoding plasmid depended neither on the deletion of the anchor domain in the gE gene nor on the animal model. Analysis of antibody isotypes produced by plasmid immunizations revealed a response slightly dominated by IgG2a. Taken together, the data indicate that a VZV subunit vaccine based on adjuvanted recombinant glycoprotein E is more promising than a nucleic acid-based vaccine strategy. As regards recgB, both vaccination approaches might be appropriate.     

Immunodominance with progenitor B cell diversity in the neutralizing antibody repertoire to influenza infection

We report striking immunodominance in the neutralizing antibody responses of major histocompatibility complex congenic mice to natural infection with influenza virus (H3N2 subtype), as deduced by sequencing the hemagglutinin (HA) genes of monoclonal antibody (mAb)-selected mutant viruses. A majority of mAb, established from individual BALB/c (H-2^d) mice, select mutant viruses containing the same single amino acid substitution in the membrane distal ectodomain, HA1 198 A→E, whereas changes at either HA1 158 G→E or HA1 198 A→E are selected for by mAb from BALB.K (H-2^k) donors. The structural basis for immunodominance, and potential diversity of progenitor B cells, was investigated by sequence analysis of H and L chain gene rearrangements in mAb specific for HA1 158 or HA1 198. No correlation was found between antibody specificity and V_H or V_L gene usage, and a minimum of three to six progenitor cells contributed to the individual's repertoire for a single antigenic site. However, in a further analysis of the HA1 158-specific antibody response of CBA/Ca (H-2^k) donors, there was highly restricted light chain gene usage. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation, particularly since there is no evident restriction in the antibody response to immunization.     

Improved assay to detect neutralizing antibody following vaccination with diluted or undiluted vaccinia (Dryvax) vaccine

The assessment of immunogenicity of a diluted vaccinia vaccine for possible widespread use of a diluted vaccine in the event of a bioterrorist attack prompted us to focus on the development of a sensitive and specific plaque reduction neutralization (PRN) assay to assess the antibody response of volunteers to a vaccinia (Dryvax) vaccine. Two incubation times, 1 h or overnight (approximately 15 h), were explored for the neutralization step of the assay. In addition, serum samples were evaluated using both sonicated and nonsonicated virus in PRN assays with 1 and 15 h of incubation. The use of the overnight incubation method resulted in the detection of antibody in two vaccinated individuals who exhibited a take, i.e., a major reaction indicative of successive vaccination as defined by the Centers for Disease Control and Prevention, but did not have a fourfold increase in antibody to vaccinia virus by the 1-h-incubation method and increased the sensitivity from 94 to 100%. In addition to the increased sensitivity of the assay, we noted a significant increase (approximately 40-fold) in the PRN titer of serum samples tested with the 15-h-incubation method. The use of sonicated virus increased the reproducibility of the virus titers and PRN titers. Forty-two percent of the samples tested using sonicated virus had a PRN titer that was fourfold higher or greater than that of nonsonicated virus in the assay. A PRN titer that was threefold higher or greater was observed in more than half (58%) of the samples using sonicated virus. Therefore, the more sensitive, specific, and reproducible plaque neutralization assay for the detection of antibody to vaccinia virus is the method using a 15-h-incubation time and freshly sonicated vaccinia virus.     

Immune responses to influenza virus infection

Influenza viruses cause annual outbreaks of respiratory tract infection with attack rates of 5-10%. This means that humans are infected repeatedly with intervals of, on average, 10-20 years. Upon each infection subjects develop innate and adaptive immune responses which aim at clearing the infection. Strain-specific antibody responses are induced, which exert selective pressure on circulating influenza viruses and which drive antigenic drift of seasonal influenza viruses, especially in the hemagglutinin molecule. This antigenic drift necessitates updating of seasonal influenza vaccines regularly in order to match the circulating strains. Upon infection also virus-specific T cell responses are induced, including CD4+ T helper cells and CD8+ cytotoxic T cells. These cells are mainly directed to conserved proteins and therefore display cross-reactivity with a variety of influenza A viruses of different subtypes. T cell mediated immunity therefore may contribute to so-called heterosubtypic immunity and may afford protection against antigenically distinct, potentially pandemic influenza viruses. At present, novel viral targets are identified that may help to develop broad-protective vaccines. Here we review the various arms of the immune response to influenza virus infections and their viral targets and discuss the possibility of developing universal vaccines. The development of such novel vaccines would imply that also new immune correlates of protection need to be established in order to facilitate assessment of vaccine efficacy.     

Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to SARS-CoV

Severe acute respiratory syndrome (SARS)-coronavirus (SARS-CoV) is the causative agent of SARS. The S protein of SARS-CoV is a major target for neutralizing antibodies (Nabs) in infected patients. We developed a neutralization assay using a recombinant vesicular stomatitis virus (VSV) bearing SARS-CoV-S protein (VSV-SARS-St19). A total of 56 serum samples collected from 22 healthcare workers in the Hanoi French Hospital during the SARS epidemic in 2003 were evaluated and compared to the conventional neutralizing assay using infectious SARS-CoV. The results of the neutralization assay using VSV-SARS-St19 pseudotype showed good correlations with those using infectious SARS-CoV. The newly developed neutralization assay was more sensitive to low antibody titers in serum samples. Thus, the VSV-SARS-St19 is a useful tool for detecting Nabs against SARS-CoV.     

Systemic and mucosal isotype-specific antibody responses in pigs to experimental influenza virus infection

The immunoglobulin isotype-specific responses in serum and at the respiratory mucosa of pigs after a primary infection with influenza virus were studied. To do this, we developed an aerosol challenge model for influenza in specified pathogen-free (SPF) pigs and isotype-specific enzyme-linked immunosorbent assays (ELISAs). Ten-week-old pigs were inoculated without anesthesia in the nostrils with an aerosol of the field isolate influenza A/swine/Neth/St. Oedenrode/96 (H3N2). The infection caused acute respiratory disease that closely resembled the disease observed in some outbreaks of influenza among finishing pigs, which were not complicated by bacterial infections. Pigs showed clinical signs characterized by fever, dyspnea, and anorexia. At necropsy on postinfection days 1 and 2, an exudative endobronchitis was observed throughout the lung. Viral antigen was present in the epithelial cells of the bronchi and bronchioli and virus was isolated from bronchioalveolar and nasal lavage fluids and from pharyngeal swabs until 5 days after infection. With the isotype-specific ELISAs, viral nucleoprotein specific immunoglobulin (Ig) M, IgG1, and IgA antibody responses were measured in serum and bronchioalveolar and nasal lavage fluids. To determine whether the antibodies were produced and secreted at the respiratory mucosa or were serum-derived, the specific activity (ie, the ratio of antibody titer to Ig concentration) was calculated for each isotype. The IgA and interestingly also a substantial part of the IgG1 antibody response in pigs upon infection with influenza virus was shown to be a mucosal response. Local production of specific IgA in the nasal mucosa, and of specific IgA and IgG1 in the lung was demonstrated. These results indicate that protective efficacy of vaccination can be improved by an immunization procedure that preferentially stimulates a mucosal immune response. The aerosol challenge model in SPF pigs and the isotype-specific ELISAs that we developed can be useful for evaluating various strategies to improve efficacy of porcine influenza vaccines and to study the immune mechanisms underlying the observed protection.     

Quantitative relationships between an influenza virus and neutralizing antibody

In this quantitative study of the interaction of influenza virus with neutralizing antibody we have determined the maximum number of antibody molecules which can bind to the haemagglutinin (HA) of native influenza A/FPV/Rostock/34 (H7N1) particles in aqueous suspension and the minimum number which is required to cause neutralization. Using radiolabelled immunoglobulins approximately one IgG molecule, whether of monoclonal or polyclonal origin, binds per HA spike under conditions of antibody saturation. In the same manner, we have determined that when infectivity is neutralized by 63% (1/e) about 70 molecules of monoclonal IgGs HC2 and HC10 were bound per virus particle and this is supported by independent evidence from electron microscopy. However, the kinetics of neutralization were single-hit or at most, under critical conditions of low temperature (4 degrees) and minimal neutralizing concentrations of antibody, two-hit. This apparent conflict is reconciled by a hypothesis which proposes that neutralization occurs only when antibody binds to certain "neutralization relevant" HA spikes which are in the minority. It is suggested that these only differ from the majority of "neutralization irrelevant" HA spikes by their transmembrane interaction with the core of the virion.     

Cytotoxic T-cell and antibody responses to influenza infection of mice.

The immune response to influenza infection was evaluated in mice using recently developed techniques to measure the induction of cytotoxic thymus-derived (T) lymphocytes and complement-dependent cytolytic antibody, locally and systemically, during primary and secondary immunization. Cytolytic antibody responses were compared to antibody titres measured by haemagglutination-inhibition (HI) and neutralization in the same samples. The development of these responses was also correlated with the titres of virus in the lung, in an attempt to further define the role of these host immune responses which can kill virus infected cells during recovery from influenza infection in vivo.     

Characterization of pseudorabies virus antibody responses in young swine after infection and vaccination by using an immunoglobulin M antibody capture enzyme-linked immunosorbent assay.

An immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MACELISA) was developed for the detection of pseudorabies virus (PRV)-specific IgM antibody in swine sera because false-positive reactions frequently occurred when sera from older swine were tested with an indirect IgM enzyme-linked immunosorbent assay. Monoclonal mouse anti-swine IgM was used as the capturing antibody, and rabbit anti-PRV hyperimmune gamma globulin was used as the indicating antibody. Sera from non-PRV-infected, experimentally infected, vaccinated and challenged, passively immune and challenged, and naturally infected swine were evaluated. The PRV MACELISA had a specificity of 95% and was as sensitive and reproducible as previously reported in direct assays. An antibody response was still detectable with the MACELISA 21 days after inoculation. The PRV MACELISA did not detect a consistent antibody response in sera from swine vaccinated with either killed-PRV or modified live-virus vaccines but did detect an antibody response in sera from passively immune pigs after challenge with virulent PRV. These results indicated that the PRV MACELISA may be useful for the rapid serodiagnosis of recent PRV infection in swine.     

Comparison of neutralizing and hemagglutination-inhibiting antibody responses for evaluating the seasonal influenza vaccine

Evaluation of the efficacy of influenza vaccines is essential for vaccine development. This study evaluated the neutralizing and hemagglutination-inhibition antibody response in subjects receiving the 2006-07 and 2007-08 seasonal influenza vaccines. ELISA-based microneutralization demonstrated a greater mean-fold increase and seroconversion rate than the hemagglutination-inhibition assay. The increase in the antibody titers against influenza H1 were higher than those against influenza H3 and influenza B, indicating that the H1 vaccine strain in the 2006-07 and 2007-08 seasons was more immunogenic. These data suggest that the neutralizing antibody response is a better measurement of influenza vaccine efficacy.     

A quantitative comet infection assay for influenza virus

The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2-6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells.     

Epitopes for broad and potent neutralizing antibody responses during chronic infection with human immunodeficiency virus type 1

Neutralizing antibody (nAb) response is sporadic and has limited potency and breadth during infection with human immunodeficiency virus type 1 (HIV-1). In rare cases, broad and potent nAbs are actually induced in vivo. Identifying specific epitopes targeted by such broad and potent nAb response is valuable in guiding the design of a prophylactic vaccine aimed to induce nAb. In this study, we have defined neutralizing epitope usage in 7 out of 17 subjects with broad and potent nAbs by using targeted mutagenesis in known neutralizing epitopes of HIV-1 glycoproteins and by using in vitro depletion of serum neutralizing activity by various recombinant HIV-1 glycoproteins. Consistent with recent reports, the CD4 binding site (CD4BS) is targeted by nAbs in vivo (4 of the 7 subjects with defined neutralizing epitopes). The new finding from this study is that epitopes in the gp120 outer domain are also targeted by nAbs in vivo (5 of the 7 subjects). The outer domain epitopes include glycan-dependent epitopes (2 subjects), conserved nonlinear epitope in the V3 region (2 subjects), and a CD4BS epitope composed mainly of the elements in the outer domain (1 subject). Importantly, we found indication for epitope poly-specificity, a dual usage of the V3 and CD4BS epitopes, in only one subject. This study provides a more complete profile of epitope usage for broad and potent nAb responses during HIV-1 infection.     

Differences in antibody responses of mice to intranasal or intraperitoneal immunization with influenza A virus and vaccination with subunit influenza vaccine

Two antigenically related but different influenza A virus strains of H3N2 subtype, A/Dunedin/ 4/73 (H3N2) (Dunedin) and A/Mississippi/1/85 (H3N2) (Mississippi), were used for intranasal (i.n.) and intraperitoneal (i.p.) immunization of mice and respective antibody responses were compared. In ELISA, using purified influenza A virus as antigen, the highest titer of antiviral antibodies was observed after a repeated i.n. infection, in which the Dunedin strain was followed by the Mississippi strain and vice versa. Similarly, in virus neutralization (VN) test, the highest titer of VN antibodies was found after a repeated i.n. infection. The subunit vaccine INFLUVAC, when administered intramuscularly (i.m.), induced only a poor antibody response as assayed by ELISA. Moreover, the INFLUVAC vaccination elicited a 100-fold lower titer of VN antibodies than the i.n. infection and an approx a 10-fold lower titer than the i.p. immunization. A repeated INFLUVAC vaccination did not lead to a significant increase of VN antibody titer. Also the antibody response to HA2gp--a conserved part of influenza hemagglutinin (HA) that might contribute to the induction of specific antiviral antibodies--was followed. Similarly to the VN antibody response, the highest HA2 antibody titer was induced after a repeated i.n. infection, whereas the lowest HA2 antibody titer was observed after a single or repeated INFLUVAC vaccination. Overall, the HA2 antibody titers remarkably well corresponded to the VN potential of the examined sera.     

A flow cytometry-based method for detecting antibody responses to murine cytomegalovirus infection

An assay based on target cells infected with green fluorescent protein labeled murine cytomegalovirus (GFP-MCMV) and dual color flow cytometry for detecting antibody to MCMV is described. After optimizing conditions for this technique, kinetics of anti-MCMV IgG antibody response was tested in susceptible (BALB/c) and resistant (C57BL/6) mouse strains following primary MCMV infection. Previously published antibody kinssetics were confirmed in susceptible mice, with peak IgG response seen approximately 8 weeks after primary infection, decreasing by 20 weeks after infection. In contrast, MCMV resistant C57BL/6 mice showed significantly lower IgG antibody responses than susceptible mice. Although several techniques have been previously described to detect murine antibody responses to MCMV, including nuclear anti-complement immunofluorescence, viral immunoblotting, complement fixation, indirect immunofluorescence, indirect hemagglutination, and enzyme-liked immunosorbent assay techniques, these techniques are all time consuming and laborious. The technique presented is a simple time efficient alternative to detect previous MCMV antibody responses in experimentally infected mice.     

Serotype-specific and cross-reactive neutralizing antibody responses in cynomolgus monkeys after infection with multiple dengue virus serotypes

Neutralizing antibody responses were examined in monkeys after dengue virus infections. In monkeys that had been infected once or twice with DENV-2, neutralizing antibody was cross-reactive with all four serotypes after secondary or tertiary infection with DENV-3. In monkeys that had been inoculated with DENV-1 and DENV-2 in the primary and secondary infections, neutralizing antibody titers did not increase after tertiary infection with DENV-3. These results indicate that antibody responses after secondary and tertiary infections with different serotypes are cross-reactive with all four serotypes, consistent with what has been observed in humans, and suggest that monkeys are useful for determining neutralizing antibody responses.