A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Immunomodulatory effects of dietary IMUNO-2865 in mice, pre-and post-vaccine challenge with parainfluenza virus 5.

Herbal remedies and nutraceuticals continue to be used as treatments for a variety of maladies ranging from joint disease to obesity. IMUNO-2865 is a natural nutraceutical supplement that has been advertised to modulate inflammation, boost cytokine activity promoting a robust immunity, but has yet to be evaluated as an adjuvant. In the present study, 4-week-old C57BL/6 female mice (n = 45) were fed 0, 5 or 50 mg/5 g tablet IMUNO-2865 (I-2865) in a tablet formulated feed. One group of mice (n = 15, 5 mice/diet) were placed on a feed diet for 14 days, while the other group of 30 mice (10 mice/diet) were placed on the diet for 28 days. Five mice from each diet group in the 28-day feeding trial were vaccinated on day 7 with a mouse recombinant parainfluenza virus to mimic viral challenge. On days 0, 14 and 28 blood samples were collected. Mice were humanely euthanized on days 14 and 28. Spleens were collected to analyze organ weight/body weight ratios, cell recovery, T cell and B cell phenotype, cell proliferation, antibody titers and cytokine production. Administration of dietary I-2865 for 14 days had no effect on murine immunity. In the 28-day dietary vaccine trial, I-2865 supplementation did not enhance vaccine response, based on vaccine antigen-specific IgG titers, nor did it alter T cell and B cell phenotype, function or cytokine response, but it did decrease splenocyte numbers in the vaccinated mice.

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.

Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response.

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (TFH) are considered important for mediating the antibody response. Most data supporting the role for TFH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of TFH-like cells in blood. We studied the activation and induction of TFH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). TFH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific TFH by LAIV was studied by flow cytometry analysis of induced TFH and CD154 expression. LAIV induced TFH proliferation, which correlated with anti-HA antibody production, and TFH were shown to be critical for the antibody response. Induction of TFH from naive T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific TFH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific TFH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, TFH, are important for the antibody response. However, data on whether TFH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing TFH In this study, we demonstrate the induction of TFH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that TFH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections.

Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats.

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse consequences of B. melitensis infection.

Helix pomatia hemocyanin - a novel bio-adjuvant for viral and bacterial antigens.

BACKGROUND: New generated subunit vaccines are characterized by increased safety and lack of side effects, however they suffer from weak immunogenicity. The adjuvants are substances that have the ability to enhance the magnitude and duration of the immune response and to increase vaccine efficacy, but the different vaccines may require diverse adjuvants. The urgent need of novel adjuvant formulations occurs, thus ensuring protective cellular and humoral responses against infectious pathogens. The hemocyanins, oxygen binding copper proteins in the hemolymph of molluscs and arthropods, are widely used as peptide carriers and vaccine adjuvants. RESULTS: In the present study we promote the hemocyanin isolated from the terrestrial gastropod Helix pomatia (HPH) as bio-adjuvant, combined with standard antigens. The purified HPH combined with influenza virus hemagglutinin intersubunit peptide (IP) or with tetanus toxoid (TT) were used for immunization. Administration of tetanus toxoid combined with HPH in mice resulted in an increased number of anti-TT IgG producing plasmocytes and induced a significant increase of B and T cell proliferation. The level of the anti-TT IgG antibodies in mice sera was comparable to the group administered with TT+Al(OH)3. An immunization of experimental animals with IP combined with H. pomatia hemocyanin led to generation of strong anti-influenza cytotoxic response. CONCLUSION: The vaccination of mice demonstrates that the HPH is acceptable as a potential bio-adjuvant for subunit vaccines and it could be used as a natural adjuvant or protein carrier.

Development and pre-clinical evaluation of two LAIV strains against potentially pandemic H2N2 influenza virus.

H2N2 Influenza A caused the Asian flu pandemic in 1957, circulated for more than 10 years and disappeared from the human population after 1968. Given that people born after 1968 are naïve to H2N2, that the virus still circulates in wild birds and that this influenza subtype has a proven pandemic track record, H2N2 is regarded as a potential pandemic threat. To prepare for an H2N2 pandemic, here we developed and tested in mice and ferrets two live attenuated influenza vaccines based on the haemagglutinins of the two different H2N2 lineages that circulated at the end of the cycle, using the well characterized A/Leningrad/134/17/57 (H2N2) master donor virus as the backbone. The vaccine strains containing the HA and NA of A/California/1/66 (clade 1) or A/Tokyo/3/67 (clade 2) showed a temperature sensitive and cold adapted phenotype and a reduced reproduction that was limited to the respiratory tract of mice, suggesting that the vaccines may be safe for use in humans. Both vaccine strains induced haemagglutination inhibition titers in mice. Vaccination abolished virus replication in the nose and lung and protected mice from weight loss after homologous and heterologous challenge with the respective donor wild type strains. In ferrets, the live attenuated vaccines induced high virus neutralizing, haemagglutination and neuraminidase inhibition titers, however; the vaccine based on the A/California/1/66 wt virus induced higher homologous and better cross-reactive antibody responses than the A/Tokyo/3/67 based vaccine. In line with this observation, was the higher virus reduction observed in the throat and nose of ferrets vaccinated with this vaccine after challenge with either of the wild type donor viruses. Moreover, both vaccines clearly reduced the infection-induced rhinitis observed in placebo-vaccinated ferrets. The results favor the vaccine based on the A/California/1/66 isolate, which will be evaluated in a clinical study.

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination.

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

Priming with AS03 A-adjuvanted H5N1 influenza vaccine improves the kinetics, magnitude and durability of the immune response after a heterologous booster vaccination: an open non-randomised extension of a double-blind randomised primary study.

An influenza vaccine with cross-immunogenic potential could play a key role in pandemic mitigation by promoting a rapid immune response to infection and/or subsequent vaccination with strains drifted from the primary vaccine strain. Here we assess the role of AS03(A) (an oil-in-water emulsion based Adjuvant System containing tocopherol) in this prime-boost concept using H5N1 as a model shift influenza antigen. In this open, non-randomised study (NCT00506350; an extension of an earlier randomised study) we assessed immunogenicity in nine groups of 35-50 volunteers aged 19-61 years following administration of AS03(A)-adjuvanted split-virion H5N1 vaccine containing 3.75mug of haemagglutinin (HA) from the A/Indonesia/5/2005(IBCDC-RG2) clade 2.1 strain. A single booster dose of vaccine was administered to four groups primed 14 months previously with different HA levels of AS03(A)-adjuvanted clade 1 A/Vietnam/1194/2004 H5N1 vaccine. Two booster doses (given 21 days apart) were administered to four groups primed 14 months previously with different HA levels of non-adjuvanted A/Vietnam/1194/2004 H5N1 vaccine and also to a control group of un-primed subjects. In individuals primed 14 months earlier with AS03(A)-adjuvanted A/Vietnam/1194/2004 vaccines, a single booster dose of AS03(A)-adjuvanted A/Indonesia/5/2005 induced rapid immune responses (licensure criteria met in 7-14 days) comparable to that observed in the un-primed control group following two doses of adjuvanted vaccine. In contrast, individuals primed with non-adjuvanted formulations exhibited minimal immune responses which, even after two doses, were unexpectedly much lower than that observed in un-primed subjects. AS03(A) enhances the initial priming effect of pandemic influenza vaccination enabling a rapid humoral response to single dose boosting with a heterologous strain at 14 months. In contrast, priming without adjuvant appears to inhibit the response to subsequent vaccination with a heterologous strain. These findings should guide the development of vaccines to combat the present influenza A/H1N1 pandemic.

Characterization of immune responses induced by intramuscular vaccination with DNA vaccines encoding measles virus hemagglutinin and/or fusion proteins.

Measles virus (MV) hemagglutinin (MV-H) and fusion (MV-F) proteins induce plaque reduction neutralizing (PRN) antibodies and cell-mediated immune responses that protect against clinical measles. DNA vaccines that encode MV-H and MV-F are being investigated as a new generation of measles vaccine to protect infants too young to receive currently licensed attenuated measles vaccines. However, it is unclear whether DNA vaccines encoding both MV-H and MV-F act synergistically to induce stronger immunity than immunization with plasmids encoding MV-H or MV-F alone. To address this question, we generated Sindbis virus-based pSINCP DNA vaccines that encode either MV-H or MV-F alone or bicistronic or fusion system vectors that encode both MV-H and MV-F (to mimic MV infection where both MV-H and MV-F proteins are expressed by the same mammalian cell). Mice immunized with DNA vaccine encoding MV-H alone developed significantly greater PRN titers than mice immunized with bicistronic constructs. Interestingly, the presence of MV-F in the bicistronic constructs stimulated serum MV-specific immunoglobulin G of reduced avidity. By contrast, mice immunized with bicistronic constructs induced equivalent or higher levels of MV-specific gamma interferon responses than mice immunized with DNA vaccine encoding MV-H alone. These data will help guide the design of DNA-based MV vaccines to be used early in life in a heterologous prime-boost strategy.

Cranberry juice constituents affect influenza virus adhesion and infectivity.

Cranberry juice contains high molecular weight materials (NDM) that inhibit bacterial adhesion to host cells as well as the co-aggregation of many oral bacteria. Because of its broad-spectrum activity, we investigated NDM's potential for inhibiting influenza virus adhesion to cells, and subsequent infectivity. Hemagglutination (HA) of red blood cells (RBC) caused by representatives of both influenza virus A subtypes (H1N1)and H3N2) and the B type was inhibited by NDM at concentrations of 125 microg/ml or lower, which is at least 20-fold lower than that usually found in cranberry juice. A dose-response effect of NDM on HA was demonstrated. The infectivity of the A and B types was significantly reduced by preincubation with NDM (250 microg/ml), as reflected by the lack of cytopathic effect on Madine-Darby canine kidney (MDCK) cells and the lack of HA activity in the media of infected cells. The effect of NDM was also tested after A or B type viruses were allowed to adsorb to and penetrate the cells. Various levels of reduction in virus tissue culture infective dose TCID50 were observed. The effect was most pronounced when NDM was added several times to the infected MDCK cells. Our cumulative findings indicate that the inhibitory effect of NDM on influenza virus adhesion and infectivity may have a therapeutic potential.

Amino-acid change on the antigenic region B1 of H3 haemagglutinin may be a trigger for the emergence of drift strain of influenza A virus.

Sera from 27 children and eight older persons, which had been collected in 1998 and 1999 and showed haemagglutination-inhibition (HI) activity against influenza A/Sydney/5/97 (H3N2) strain, were characterized with a binding assay using chimeric haemagglutinin (HA) proteins between A/Aichi/2/68 (A/AI/68) and A/Sydney/5/97 (A/SD/97) strains. Sera from the young children had a tendency to recognize only the antigenic site B1 of the HA1 region. On the other hand, sera of the older individuals were fully reactive to all antigenic sites of HA1 except antigenic site D. Recent epidemic strains, A/Panama/2007/99 (A/PM/99)-like viruses have differences in amino acids in antigenic sites A, C, and B2 but not B1. However, human antisera obtained even from young children had HI activity to Panama-like viruses. The limited epidemic of A/PM/99-like viruses may have been due to the existence of antibody against B1, which had been produced in response to infection by the A/SD/97-like viruses.

Neutralizing immunogenicity of transgenic carrot (Daucus carota L.)-derived measles virus hemagglutinin.

Although edible vaccines seem to be feasible, antigens of human pathogens have mostly been expressed in plants that are not attractive for human consumption (such as potatoes) unless they are cooked. Boiling may reduce the immunogenicity of many antigens. More recently, the technology to transform fruit and vegetable plants have become perfected. We transformed carrot plants with Agrobacterium tumefaciens to generate plants (which can be eaten raw) transgenic for an immunodominant antigen of the measles virus, a major pathogen in man. The hemagglutinin (H) glycoprotein is the principle target of neutralizing and protective antibodies against measles. Copy numbers of the H transgene were verified by Southern blot and specific transcription was confirmed by RT-PCR. The H protein was detected by western blot in the membrane fraction of transformed carrot plants. The recombinant protein seemed to have a 8% lower molecular weight than the viral protein. Although this suggests a different glycosylation pattern, proper folding of the transgenic protein was confirmed by conformational-dependent monoclonal antibodies. Immunization of mice with leaf or root extracts induced high titres of IgG1 and IgG2a antibodies that cross-reacted strongly with the measles virus and neutralized the virus in vitro. These results demonstrate that transgenic carrot plants can be used as an efficient expression system to produce highly immunogenic viral antigens. Our study may pave the way towards an edible vaccine against measles which could be complementary to the current live-attenuated vaccine.

C3d enhancement of neutralizing antibodies to measles hemagglutinin.

Measles remains a major cause of worldwide infant mortality despite the use of current live attenuated vaccines. New approaches to measles virus (MV) vaccine development are critical to interrupt the spread of MV. In this study, we report the results using a DNA vaccine expressing a fusion of the measles hemagglutinin (H) protein and the complement component, C3d, to enhance the titers of neutralizing antibody. Plasmids were generated that expressed a secreted (s) form of H and the same form fused to three tandem copies of the murine homologue of C3d (sH-3C3d). Analysis of titers of the antibody raised in vaccinated mice indicated that immunizations with the DNA expressing sH-3C3d had higher titers of anti-H antibodies compared to serum from mice vaccinated with DNA expressing sH only. In addition, sH-3C3d elicited higher neutralizing antibody titers that inhibited MV induced plaque formation.

Antigen-specific elimination of T cells induced by oligomerized hemagglutinin (HA) 306-318.

In a previous study we reported that oligomerized T cell epitopes "superactivated" CD4+ T cells. These oligomers, consisting of 12-16 copies of a peptide epitope derived from the hemagglutinin protein of influenza virus (HA306-318), induced a specific T cell response in amounts as little as 5 pg/ml. We now show that the improved antigenicity of these multimerized epitopes can also be utilized to induce "high zone tolerance". Tolerization, similar to activation, occurred at about 3 logs lower concentration of oligomer than of peptide. HA306-318-specific T cell cultures became nonresponsive to stimulation with peptide after incubation with 0.5-5 microg/ml HA306-318 12-mer. The nonresponsiveness was accompanied by a drastic down-regulation of the TCR and by T cell elimination by apoptotic cell death. In contrast, stimulation with peptide even at 50 microg/ml led to temporary induction of anergy. Consequently, induction of tolerance with the oligomer was permanent and no recovery of the cultures was seen in recall experiments 12-14 days after high zone exposure to the 12-mer.

Calcitriol (1,25-dihydroxy-vitamin D3) coadministered with influenza vaccine does not enhance humoral immunity in human volunteers.

Calcitriol, also known as 1,25-dihydroxy-vitamin D3, is a steroid hormone that has been shown to have effects on cytokine production and lymphocyte proliferation. Coadministration of calcitriol with trivalent influenza vaccine in mice enhanced both mucosal and systemic antibody responses. We studied the effects of calcitriol coadministered with a commercially available influenza vaccine in 175 human volunteers in this double-blind, placebo controlled clinical trial. Subjects that received calcitriol experienced more pain at the injection site compared with placebo recipients. No significant differences in hemagglutination inhibition titers against H1N1, H3N2, or influenza B antigens were detected at 1 or 3 months postvaccination. We conclude that coadministration of 1.0 microg of calcitriol at a site adjacent to influenza vaccination does not enhance humoral immunity in human volunteers.

Crystallization and preliminary X-ray diffraction studies of complexes between an influenza hemagglutinin and Fab fragments of two different monoclonal antibodies.

Fab fragments from two different monoclonal antibodies (BH151 and HC45) which bind to the same antigenic region of the influenza hemagglutinin were crystallized as complexes with the hemagglutinin. The complexes crystallize in PEG 600, pH 6.0, and PEG 2000, pH 8.5, respectively. Both crystals belong to space group P321, with very similar unit cell dimensions.

Comparison of the effects of five adjuvants on the antibody response to influenza virus antigen in guinea pigs.

Five adjuvants were tested for their effect on the immune response in guinea pigs to the hemagglutinin antigen of influenza virus strain B/Panama. Vaccines containing 924 micrograms of hemagglutinin antigen/ml were prepared at high and low doses of Freund's complete and incomplete adjuvants, Syntex adjuvant, RIBI's adjuvant, TiterMax adjuvant, and aluminum phosphate adjuvant. Responses to these vaccines were compared with those to a control vaccine containing influenza virus B/Panama hemagglutinin antigen and saline. On day 28, vaccines containing the following adjuvant doses had significantly higher titers than the titer for the control: Freund adjuvants at high and low doses, RIBI at high dose, TiterMax at high and low doses, and aluminum phosphate at high dose. On day 42, vaccines containing the following adjuvant doses had significantly higher titers than that for the control: Freund adjuvants at high and low doses, RIBI at high dose, TiterMax at high dose, and aluminum phosphate at high dose. Freund adjuvants at high and low doses, RIBI adjuvant at high dose, and aluminum phosphate at high dose caused significantly greater swelling at the inoculation site than did the control vaccine. TiterMax adjuvant at high and low doses, and aluminum phosphate at low dose caused minor swelling at the inoculation site, but it was not significantly different from the swelling caused by the control vaccine. Syntex adjuvant at high and low doses, RIBI at low dose, and control (saline/antigen) at high and low doses caused no swelling after inoculation. Overall, the high dose of adjuvants caused greater tissue swelling than did the low dose of adjuvants.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopy of antibody complexes of influenza virus haemagglutinin in the fusion pH conformation.

Activation of the membrane fusion potential of influenza haemagglutinin (HA) at endosomal pH requires changes in its structure. X-ray analysis of TBHA2, a proteolytic fragment of HA in the fusion pH conformation, indicates that at the pH of fusion the 'fusion peptide' is displaced by > 10 nm from its location in the native structure to the tip of an 11 nm triple-stranded coiled coil, and that the formation of this structure involves extensive re-folding or reorganization of HA. Here we examine the structure of TBHA2 with the electron microscope and compare it with the fusion pH structure of HA2 in virosomes, HA2 in aggregates formed at fusion pH by the soluble, bromelain-released ectodomain BHA and HA2 in liposomes with which BHA associates at fusion pH. We have oriented each HA2 preparation for comparison, using site-specific monoclonal antibodies. We conclude that the structural changes in membrane-anchored and soluble HA preparations at the pH of fusion appear to be the same; that in the absence of a target membrane, the 'fusion peptide' of HA in virosomes associates with the virosome membrane so that HA2 is membrane bound at both N- and C-termini, which implies that inversion of the re-folded HA can occur; and that the structural changes observed by X-ray analysis do not result from the proteolytic digestions used in the preparation of TBHA2.

Microvariation creates significant functional differences in the DR3 molecules.

Two DR3 molecules differ by four amino acids whose side chains point into the DR antigen-binding groove. To begin to assess the role of microvariation on DR3 function, DRB1*0302 residues were replaced with DRB1*0301 residues at beta-chain positions 26, 47, 86, and 47 plus 86. Murine fibroblast cell lines expressing DR(alpha, beta 1*0301), DR(alpha, beta 1*0302), and the four mutant 0302 molecules were examined for alloproliferative DR(alpha, beta 1*0302)-specific TLC stimulation and peptide binding. Changing position 26 had the most profound effect on T-cell recognition (seven of nine TLCs did not respond). Two TLCs did not respond to the mutant 0302V86 molecule and four TLCs that did respond to this mutant lost responsiveness when positions 47 and 86 were mutated together. These data suggest that each of these variant residues, including position 47, influence T-cell recognition. Surprisingly, none of the mutations had an effect on the absolute binding of HA 307-319 (DR[alpha, beta 1*0302] specific) and HSP 3-13 (DR[alpha, beta 1*0301] specific); however, the mutant 0302 molecules changed at position 86 (glycine to valine) consistently bound HA 307-319 at significantly higher levels than DR(alpha, beta 1*0302). These data for position 86 are in contrast to other DR molecules and indicate that peptide contact residues for a specific DR molecule cannot be predicted based on binding results obtained with other DR molecules. These data suggest that each of these variant groove residues, although not accessible to the TCR, contribute to the significant functional differences between the DR3 microvariants through subtle influences on the DR3-peptide complex.