Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H3N2) virus, together with cholera toxin B subunit as an adjuvant, were examined for protection against PR8 infection; PR8 HA and A/Yamagata HA immunization conferred complete protection, while A/Fukuoka HA immunization failed to confer protection. In parallel with protection, PR8 HA-, A/Yamagata HA-, and A/Fukuoka HA-immunized mice produced a high, a moderate and a low level of PR8 HA-reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross-reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA-immunized mice, which contained the HA-specific IgA corresponding to the amount detected in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from A/Yamagata HA-immunized mice also afforded cross-protection against PR8 infection, whereas the IgA from A/Fukuoka HA-immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA-reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza A virus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.     

Lower respiratory tract infection caused by respiratory syncytial virus in infants: the role played by specific antibodies

INTRODUCTION: Respiratory syncytial virus (RSV) is a major etiological agent of lower respiratory tract infection in infants. Genotypes of this virus and the role of the infants' serum antibodies have yet to be fully clarified. This knowledge is important for the development of effective therapeutic and prophylactic measures. OBJECTIVES: To evaluate the types and genotypes of RSV causing respiratory tract infection in infants, to analyze the association of subtype-specific serum antibodies with the occurrence of infection and to evaluate the presence of subtype-specific antibodies in the infants' mothers and their association with the profile of the childrens' serum antibodies. METHODS: This was a prospective study on infants hospitalized with respiratory infection. Nasopharyngeal secretions were collected for viral investigation using indirect immunofluorescence and viral culture and blood was collected to test for antibodies using the Luminex Multiplex system. RESULTS: 192 infants were evaluated, with 60.9% having RSV (73.5%- A and 20.5% B). Six genotypes of the virus were identified: A5, A2, B3, B5, A7 and B4. The seroprevalence of the subtype-specific serum antibodies was high. The presence and levels of subtype-specific antibodies were similar, irrespective of the presence of infection or the viral type or genotype. The mothers' antibody profiles were similar to their infants'. CONCLUSIONS: Although the prevalence of subtype-specific antibodies was elevated, these antibodies did not provide protection independently of virus type/genotype. The similarity in the profiles of subtype-specific antibodies presented by the mothers and their children was consistent with transplacental passage.     

Production of monoclonal antibodies specific to theophylline-treated lymphocytes

The T11 molecule is reported to play a key role in T lymphocytes activation. Moreover, theophylline is known to modify the functional properties of T lymphocytes probably inducing early changes in T11 molecule during T cell activation. Aim of our work was to clarify the effect on T lymphocyte surface after in vitro treatment with theophylline. In this paper, we describe the production and the functional properties of several monoclonal antibodies obtained by immunizing Balb/c mice with theophylline treated cells. Some of the monoclonal antibodies reacted only with the theophylline-treated lymphocytes and showed a promitogenic activity, enhancing the expression of T activated cell antigen. These monoclonal antibodies seem to demonstrate the existence of a membrane molecule which appears on lymphocytes surface after a trigger signal occurring in the early stages of T cell activation likely related to the T11 dependent alternative pathway.     

Production of IgA monoclonal antibodies against influenza A virus

Nineteen IgA monoclonal antibodies against influenza A virus X-31 were obtained following intranasal infection of mice with influenza A virus X-31. It was demonstrated that specificities of IgA monoclonal antibodies are similar to those of IgG monoclonal antibodies. These IgA antibodies might be useful for the study of mucosal immunity against influenza A viruses. Infection may be an easier and better way of producing monoclonal antibodies against some viral agents.     

T lymphocytes as an indicator in influenza vaccination, influenza and acute respiratory diseases.

Significant differences in T-lymphocyte counts in the peripheral blood of normal subject and volunteers vaccinated with live and inactivated influenza vaccines as well as patients with influenza and viral acute respiratory diseases (ARD) were demonstrated. A laboratory test using T lymphocytes was proposed for the evaluation of the safety of live influenza vaccine.     

Production of highly cross-reactive hemagglutination-inhibiting influenza antibodies in ferrets.

Ferrets were sequentially infected at time intervals of 3 weeks with different influenza virus A strains. It was found that secondary infection can result in the appearance of antibodies highly cross-reacting with a virus strain closely related to the strain of first infection. Such highly cross-reacting antibodies were designated as HCR antibodies. Evidence is presented that HCR antibodies were not antineuraminidase antibodies and, in addition, were not specifically oriented to the strain with which the crossing was observed. When using time intervals of 3 weeks between infections, no increase after secondary infection of antibodies oriented to the strain of first infection was recorded. However, when time intervals of 4 to 5 months between infections were used, secondary infections resulted in an increase of antibodies oriented to the strain of first infection ("original antigenic sin") but not in the appearance of HCR antibodies. In addition, antibodies combining specifically with both infecting strains, designated as doubly specific antibodies, were found. Thus, the conclusion was reached that the original antigenic sin phenomenon and the appearance of HCR antibodies are mutually exclusive events.     

Inactivation of influenza virus by human lymphocytes

The interaction of influenza virus and human lymphoid cells has been studied by electron microscopy. The results suggest that virus is inactivated by lymphocytes after uptake of virus into cytoplasmic vacuoles. Over a period of 24 hr, the virus particles are processed by the cell, the vast majority becoming undetectable by electron microscopy.     

Improved detection of human influenza A and B viruses in respiratory tract specimens by hemi-nested PCR

RT-PCR is the most sensitive assay for diagnosis of influenza, due to enhanced rapidity and sensitivity as compared to classical methods. Hemi-nested RT-PCR was developed, targeting NP gene for influenza A and NS gene for influenza B, based on a previous single round RT-PCR method. The new method was compared with the previous technique for analytical sensitivity and specificity, and was applied to clinical samples from the lower and upper respiratory tract. The analytical sensitivity of hemi-nested RT-PCR was 10 (influenza A) and 4 times (influenza B) higher than the previous method. A high specificity of the new hemi-nested RT-PCR assay was observed by using whole respiratory viruses. When applied to lower respiratory tract specimens, the new method showed an increased rate of positivity as compared to the previous technique (9.3% versus 0.7% for influenza A, and 0.9% versus 0.2% for influenza B). Screening of upper respiratory tract samples collected during the seasonal 2005-2006 outbreak indicated 26.4% and 5.8% positivity for influenza A and B, respectively. The results were confirmed by sequence analysis: apart from influenza B, both influenza A subtypes H3N2 and H1N1, associated with the seasonal outbreak, were detected.     

A study of possibility to predict the sensitivity of different respiratory tract divisions to influenza

To predict a potential value of a viral ID50 for a macro-organism of interest (e.g. humans), it is necessary to determine in vitro two parameters of the interaction of the virus with susceptible cells of the host, i.e. the probability of the virus' productive absorption on a susceptible cell and the average virus yield per cell. A different macroorganism (a model animal) and primary cells obtained from it can be used to determine the value of a scale factor, which accounts for the difference between the values of the probability of the virus' absorption measured in vivo and in vitro. An original mathematical model is used to convert the above-mentioned data to ID50 for the macroorganism of interest. It was shown that the method of cultivating influenza virus (A/ Aichi/2/68) in primary suspension culture of respiratory tract cells of rats and two breeds of mice may be used to estimate potential human susceptibility to novel influenza viruses. This work was sponsored by DAPRA, USA, and performed under the contract 450p to the International Science and Technology Center, Moscow.     

Functionally distinct T cells in three compartments of the respiratory tract after influenza virus infection

This study aimed to resolve, firstly, whether T cell responses induced in one tissue site are similar to those induced by the same antigen in another site and, secondly, whether influenza virus infection induces one predominant type of T cell response locally in the respiratory tract. To address these questions, T cell responses in three compartments of the respiratory tract were compared after infection of mice with a sublethal dose of influenza virus: the draining mediastinal lymph nodes (MLN), the lung parenchyma and the airways. Each compartment harbored a T cell response substantially different from that found at the other sites. A preferential accumulation of ex vivo-cytolytic CD8⁺ T cells was found in the airways (CD4/CD8 ratio 1:2) and to a lesser extent in the lung parenchyma (CD4/CD8 ratio 1:1). T cells from both compartments expressed high levels of various cytokine mRNA, but showed differences in their respective expression pattern, with those from lung tissue showing particularly high levels of IFN-γ mRNA. The response in the draining lymph nodes, on the other hand, was dominated by CD4⁺ T cells (CD4/CD8 ratio 2:1) with a higher proliferative capacity (after TCR/CD3 cross-linking) and which provided better B cell help in vitro than CD4⁺ T cells isolated from lung tissue. T cells from MLN expressed mRNA for a variety of cytokines with only low levels of IFN-γ mRNA and they showed no CTL activity ex vivo. These functional differences were not due to differences in the kinetics of the response, or to the higher frequencies of activated T cells in lung tissue and airways compared to MLN, since the differences remained when cell-sorter-purified activated (CD18^hi, CD44^hi) T cells from MLN and lung tissue were compared in a time-course study. Taken together, these findings indicate that pathogens such as influenza virus induce a heterogenous set of T cell responses in different tissue sites affected by the infection.     

Production and diagnostic application of monoclonal antibodies against influenza virus H5

Nine monoclonal antibodies (mAbs) against avian influenza virus (AI) H5 subtype from mice immunized with inactivated virus H5N1 (A/Turkey/ON/6213/66) were produced. Upon testing, the results indicated that the binding epitopes of eight out of the nine mAbs were conformational, while one mAb (#7) reacted with denatured H5N1 only. Two mAbs #10 and #11 reacted with all of the thirteen H5 strains tested indicating that the binding epitopes of these mAbs were conserved among these H5 subtypes.Possible applications of these mAbs in rapid tests for H5 antigen were explored. Double antibody sandwich (DAS) ELISAs were developed using two selected mAbs #10 and #11. This DAS ELISA detects specific H5 viruses and is able to identify all thirteen H5 strains tested. Three mAbs showed reactivity with AI H5 antigen for both immunofluorescence (IF) and immunohistochemistry. A cELISA used to screen chickens that had been infected with an H5 virus was developed with mAb #9 and recombinant H5 antigen. The sera from chickens that have been infected with an H5N1 virus were examined using the cELISA. 80% of the sera from H5 infected chickens showed a positive H5 specific antibody response at 7 days post-infection (dpi) and remained positive until the end of the experiment on day 30 (>40% inhibition). This panel of the AI H5 specific mAbs is valuable for the development of various immunoassays.     

Progression of influenza viral infection through the murine respiratory tract: the protective role of sleep deprivation

Sleep deprivation is reported to have both beneficial and harmful effects upon host defenses. In the work reported herein, we address the effects of sleep deprivation on the mucosal anti-influenza defenses of both immune and nonimmune BALB/c mice. Sleep deprivation does not depress existing mucosal antiviral defenses in the respiratory tracts of BALB/c mice; in fact, it may actually be beneficial. Nasal mucosal immunity is not adversely affected in immune mice by sleep deprivation. In nonimmune mice, sleep deprivation slows or prevents the progress of nasal influenza viral infection down the trachea into the lungs. By 72 hours post-infection, 12 of 12 control mice shed virus into bronchioalveolar lavages (BAL) while only 2 of 12 sleep deprived mice shed virus (p<0.001). BAL levels of IL-1beta and interferon alpha were increased in sleep deprived animals, suggesting that sleep deprivation may exert its beneficial effects on the respiratory tract by upregulating the production of antiviral cytokines.     

The delivery site of a monovalent influenza vaccine within the respiratory tract impacts on the immune response

Pulmonary vaccination is a promising immunization route. However, there still remains a crucial need to characterize the different parameters affecting the efficacy of inhaled vaccination. This study aimed at assessing the impact of antigen distribution within the respiratory tract on the immune response to a monovalent A/Panama/2007/99 H3N2 influenza split virus vaccine administered to BALB/c mice. Varying the administration technique allowed the targeting of the vaccine to different sites of the mouse respiratory tract, i.e. the nasal cavity, the upper or central airways, or the deep lung. This targeting was verified by using ovalbumin as a tracer compound. The immune responses generated following influenza vaccine administration to the different respiratory tract sites were compared to each other and to those elicited by intramuscular and peroral intragastric immunization. Delivery of the vaccine to the different respiratory regions generated systemic, local and cellular virus-specific immune responses, which increased with the depth of vaccine deposition, culminating in deep-lung vaccination. The latter induced virus-specific serum immunoglobulin G and neutralizing antibody titres as elevated as intramuscular vaccination, whereas the production of mucosal secretory immunoglobulin A was significantly superior in deep-lung-vaccinated animals. The analysis of cytokines secreted by mononuclear cells during an in vitro recall response indicated that deep-lung vaccination induced a local shift of the cellular immune response towards a T helper type 1 phenotype as compared to intramuscular vaccination. In conclusion, antigen distribution within the respiratory tract has a major effect on the immune response, with the deep lung as the best target for inhaled influenza vaccination.