Development of a Skin Test for Bovine Tuberculosis for Differentiating Infected from Vaccinated Animals

The tuberculin skin test has been used for the diagnosis of bovine and human tuberculosis (TB) for over a hundred years. However, the specificity of the test is compromised by vaccination with the Mycobacterium bovis-derived vaccine strain bacille Calmette-Guérin (BCG). Since current promising vaccines against bovine TB are based on heterologous prime-boost combinations that include BCG, there is a need for diagnostic tests for differentiating infected from vaccinated animals (DIVA). The application of antigens such as ESAT-6 and CFP-10 for DIVA has so far been realized largely through their application in the blood-based gamma interferon release assay. In the current study, we have reassessed the potential of such antigens as skin test reagents for DIVA in cattle. A cocktail of the Mycobacterium tuberculosis complex recombinant protein antigens ESAT-6, CFP-10, MPB70, and MPB83 elicited delayed-type hypersensitivity (DTH) skin test responses in 78% of naturally infected tuberculin-positive cattle. Importantly, this cocktail induced no skin responses in BCG-vaccinated cattle despite them being sensitized for strong tuberculin responses. Further optimization of skin test antigen combinations identified that the inclusion of Rv3615c (Mb3645c) enhanced skin test sensitivity in naturally infected cattle without compromising specificity. In addition, we demonstrate for the first time the utility of synthetic peptides as promising skin test antigens for bovine TB for DIVA. Our data provide a promising basis for the future development of skin tests for DIVA with practical relevance for TB diagnosis in both veterinary and clinical settings.Bovine tuberculosis (TB) is a disease of economic and zoonotic importance caused by infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. The primary diagnostic test used for the control and surveillance of bovine TB is the tuberculin skin test, a test that has remained in the forefront of TB diagnosis for both humans and cattle for over 100 years. The development of the test arose following the preparation of the first “tuberculin” by Robert Koch in 1890 (9). While Koch''s tuberculin failed to live up to its initial claims of having curative properties, its diagnostic potential was quickly realized. Interestingly, its potential to diagnose tuberculous infection was first demonstrated in cattle prior to its widespread use in people (19). The two most common formats of the test used in cattle are the caudal fold test and the single intradermal comparative cervical tuberculin (SICCT) test (13). Both test formats use a purified protein derivative (PPD) tuberculin prepared from a culture of M. bovis (PPD-B) as the primary diagnostic antigen. Additionally, the SICCT test includes the use of a Mycobacterium avium-derived PPD (PPD-A) to provide a measure of environmental sensitization. It is the more specific of the two tests (14, 20) and is therefore the adopted test format in the United Kingdom.Bovine TB continues to be a significant and ongoing problem in the United Kingdom (www.defra.gov.uk/foodfarm/farmanimal/diseases/atoz/tb/index.htm). Cattle vaccination has been identified as one of the most promising long-term United Kingdom control strategies (10), and the development of an efficacious vaccine continues to be a research priority. Currently, promising vaccines against bovine TB are based on heterologous prime-boost combinations that include the live attenuated M. bovis vaccine strain bacille Calmette-Guérin (BCG) as one of their components (8). However, as for humans, the vaccination of cattle with BCG compromises the specificity of the tuberculin skin test since PPD contains cross- reactive antigens shared by both pathogenic and vaccine strains (4, 5, 26). Therefore, the development of diagnostic tests for differentiating vaccinated from infected animals (DIVA) is an essential prerequisite to allow the inclusion of BCG-based vaccination as part of bovine TB control strategies.Current front-line reagents for DIVA for TB diagnosis are the M. tuberculosis complex antigens ESAT-6 and CFP-10. They are strongly recognized in TB-infected cattle and humans (6, 15, 22, 24, 25), yet since their genes are encoded in region of difference 1 (RD1), which has been deleted from all BCG strains (12), they do not elicit a response in BCG-vaccinated hosts in the absence of infection (2, 6, 22, 24, 25). The practical application of such reagents for DIVA has so far been realized largely through their use in blood-based gamma interferon (IFN-γ) release assays (IGRAs). Given the high level of familiarity and widespread application of the tuberculin skin test by veterinarians and clinicians, a skin test format for DIVA would provide a valuable additional test platform. This might especially be the case where the logistics of access to laboratory-based resources are problematic. It is also notable that in recent years, there has also been renewed interest in a skin test-based test for DIVA for human TB, with several reports demonstrating the skin test potential of ESAT-6 (1, 3, 28).The purpose of the current study was to assess the potential of defined M. bovis antigens as skin test reagents for diagnosing bovine TB. We have investigated and optimized combinations of define M. bovis antigens with an emphasis on assessing their performance as sensitive and practical diagnostic tools for DIVA for use in the field.     

Generation and evaluation of an H9N1 influenza vaccine derived by reverse genetics that allows utilization of a DIVA strategy for control of H9N2 avian influenza

H9N2 avian influenza viruses have circulated widely in domestic poultry around the world, and their outbreaks have resulted in heavy morbidity and mortality. In addition, H9N2 avian influenza viruses were transmitted directly from birds to humans in Hong Kong and mainland China during 1998 and 2003, which prompted the public health authorities to seek protective strategies to control H9N2 influenza viruses. In this study, we attempted to develop a DIVA (differentiating infected and vaccinated animals) strategy for H9N2 avian influenza viruses. This strategy does not interfere with serological monitoring and allows effective control of H9N2 avian influenza. We generated a reassortant H9N1 influenza vaccine strain by reverse genetics and employed an enzyme-linked immunosorbent assay (ELISA) with a truncated N1 antigen expressed in E. coli to differentiate between vaccinated and naturally infected animals. Immunization of BALB/c mice with the inactivated reassortant H9N1 vaccine conferred protection against lethal challenge with H9N2 viruses. Meanwhile, the ELISA can be used to distinguish between vaccination and natural infection quickly and easily. Therefore, this study has opened up a new avenue for the control of H9N2 avian influenza.     

Virus-Like Particle Vaccine Confers Protection against a Lethal Newcastle Disease Virus Challenge in Chickens and Allows a Strategy of Differentiating Infected from Vaccinated Animals

In this study, we developed Newcastle disease virus (NDV) virus-like particles (VLPs) expressing NDV fusion (F) protein along with influenza virus matrix 1 (M1) protein using the insect cell expression system. Specific-pathogen-free chickens were immunized with oil emulsion NDV VLP vaccines containing increasing dosages of VLPs (0.4, 2, 10, or 50 μg of VLPs/0.5-ml dose). Three weeks after immunization, the immunogenicity of the NDV VLP vaccines was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit, and a lethal challenge using a highly virulent NDV strain was performed to evaluate the protective efficacy of the NDV VLP vaccines. NDV VLP vaccines elicited anti-NDV antibodies and provided protection against a lethal challenge in a dose-dependent manner. Although the VLP vaccines containing 0.4 and 2 μg of VLPs failed to achieve high levels of protection, a single immunization with NDV VLP vaccine containing 10 or 50 μg could fully protect chickens from a lethal challenge and greatly reduced challenge virus shedding. Furthermore, we could easily differentiate infected from vaccinated animals (DIVA) using the hemagglutination inhibition (HI) test. These results strongly suggest that utilization of NDV VLP vaccine in poultry species may be a promising strategy for the better control of NDV.     

Molecular cloning and analysis of the N5 neuraminidase subtype from an avian influenza virus

The neuraminidase (NA) gene from the prototype N5 influenza virus, A/Shearwater/Australia/72, has been cloned and completely sequenced. An open reading frame of 1404 bp (468 amino acids) is flanked by 20-bp 5'- and 31-bp 3'-untranslated regions. The deduced amino acid sequence of the N5 gene was compared with sequences from N2, N1, N7, N8, and N9 subtypes. One hundred thirteen amino acid residues (24%) are completely conserved across subtypes and include active site residues, cysteines, potential glycosylation sites, and certain glycines which suggests that these subtypes share a common ancestor and adopt the same 3-D conformation. Three groups can be assigned from amino acid homologies: (i) N5, N8, N1; (ii) N7, N9; and (iii) N2 where the percentage identity within groups is 55-68% and between groups is 40-46%, the N5-N8 pair bearing the closest identity (68%). Phylogenetic analysis suggests that these groups diverged concurrently.     

Purification,Stability, and Immunogenicity Analyses of Five Bluetongue Virus Proteins for Use in Development of a Subunit Vaccine That Allows Differentiation of Infected from Vaccinated Animals

Bluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus or Escherichia coli expression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or −80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n = 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.     

Development and validation of an anti-N3 indirect immunofluorescent antibody test to be used as a companion diagnostic test in the framework of a "DIVA" vaccination strategy for avian influenza infections in poultry.

Avian influenza (AI) infections have become of growing importance both for animal and human health. Vaccination has become a recommended tool to support eradication efforts and limit the economic losses caused by this disease. The "DIVA" system, using a vaccine containing a heterologous neuraminidase to the field virus, has been shown to be an effective tool in increasing the resistance of birds to field challenge, preventing clinical signs and reducing viral shedding in the environment. The companion diagnostic test to the vaccine, however, has been only partially validated in the field against one subtype of neuraminidase (N1). The present paper presents the results of a full laboratory and field validation of the diagnostic test developed to detect antibodies to the N3 subtype of AI in vaccinated and unvaccinated chickens and turkeys. Antibody kinetic studies conducted in the laboratory have shown that antibodies to the N protein may be detected earlier than antibodies to the haemagglutinin. The data derived from this extensive validation trial indicate the excellent capability of this assay in detecting the presence of active AI infection at an early stage in both unvaccinated and vaccinated birds and the lack of interference with vaccine-induced antibodies.     

含有H5N1 NA基因重组腺病毒疫苗在小鼠体内的免疫效果研究

目的 考察含有禽流感病毒A/Anhui/1/2005(H5N1)优化型NA基因的重组腺病毒在BALB/c小鼠体内的免疫效果,并筛选合适的免疫剂量.方法 重组病毒以肌内注射方式在第0周和第4周免疫BALB/c小鼠两次,低、中、高组的免疫剂量分别是10~5、10~7和10~9 TCID_(50)/次,第5周时分别使用神经氨酸酶活性抑制试验和EHSpot实验来检测和比较疫苗的体液和细胞免疫效果.结果 重组病毒免疫后的小鼠均可检测出针对NA抗原的特异性体液和细胞免疫反应,并且免疫效果与免疫剂量呈正相关,10~7 TCID_(50)/只/次是合适的免疫剂量.另外,从包含NA全长氨基酸残基的合成肽库中筛选到两个能够刺激BALB/c小鼠T淋巴细胞分泌IFN-γ的表位,即NA_(109-124):CRTFFLTQGALLNDKH和NA_(182-199):AVAVLKYNGIITIKSW.结论 含有优化型NA基因的重组腺病毒疫苗能够诱导BALB/c小鼠同时产生NA抗原特异性的体液和细胞免疫反应,是较好的H5N1流感病毒候选疫苗株,值得进一步深入研究.     

Development of a mucosal vaccine for influenza viruses: preparation for a potential influenza pandemic

Highly pathogenic avian H5N1 influenza A virus has caused influenza outbreaks in poultry and migratory birds in Southeast Asia, Africa and Europe, and there is concern that it could cause a new pandemic. This fear of an emerging pandemic of a new influenza strain underscores the urgency of preparing effective vaccines to meet the pandemic. One way to mitigate current concerns is to develop an influenza vaccine that is fully functional against drift influenza viruses. In our current situation, in which we cannot predict which strain will cause a pandemic, cross-protective immunity using potential and novel mucosal vaccines plays a particularly important role in preventing the spread of highly pathogenic influenza virus.     

Characterization of the hemagglutinin and neuraminidase genes of recent influenza virus isolates from different avian species in Thailand

The hemagglutinin (HA) and neuraminidase (NA) genes of eight influenza A virus (H5N1) isolates obtained from various avian species in Thailand in 2003-2004 have been characterized in comparison with the Thai isolate A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04(H5N1). Phylogenetic analyses of both genes revealed that all the eight avian isolates were closely related to the A/Chicken/Nakorn-Pathom/Thailand/CU-K2/ 04(H5N1). The amino acid sequence of the HA cleavage site revealed a common characteristic of a highly pathogenic virus strain. Moreover, a deletion of 20 amino acids in the NA stalk region was detected in all Thai isolates in contrast to the H5N1 strain that had caused outbreaks in eastern Asia in 1996-1997 and 2000-2001.     

Strategies for improving the efficacy of a H6 subtype avian influenza DNA vaccine in chickens

A low-pathogenicity avian influenza H6N2 virus was used to investigate approaches to improve DNA vaccine efficacy. The viral hemagglutinin (HA) gene or its chicken biased HA gene, incorporating a Kozak sequence, was cloned into a pCAGGS vector to produce the pCAG-HAk and pCAG-optiHAk constructs. Following two intramuscular injections, the seroconversion rate in vaccinated chickens with 10, 100 or 300 μg pCAG-HAk were 87.5%, 75% and 75%, respectively. The profile of H6 hemagglutination inhibition (HI) antibodies induced by different doses of pCAG-HAk during the 8-week study period was similar. The HI titer rose significantly in the three different dose groups following the booster and reached a plateau 2-3 weeks post-booster. In a single dose vaccination group with 100 μg pCAG-HAk, a maximum seroconversion rate reached 53.3% at 5 weeks post-vaccination. The earliest time of seroconversion appeared two weeks after DNA immunization. Following two electroporation (EP) vaccinations with 100 μg pCAG-HAk, all birds seroconverted and the HI antibody titers were significantly higher than those using intramuscular immunization, suggesting that EP was more efficient than intramuscular delivery of the DNA vaccines. In comparison, chickens immunized with 10 or 100 μg pCAG-optiHAk showed 37.5% and 87.5% seroconversion rates, respectively, at 3 weeks following the booster. The pCAG-HAk was not significantly different from the pCAG-optiHAk in either the seroconversion rate or H6 HI titer, suggesting that the codon-optimized HA DNA vaccine did not achieve significantly better immunogenicity than the pCAG-HAk vaccine.     

Development of a recombinant ELISA using yeast (Pichia pastoris)-expressed polypeptides for detection of antibodies against avian influenza A subtype H5

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.     

The control of H5 or H7 mildly pathogenic avian influenza: A role for inactivated vaccine

Biosecurity is the first line of defence in the prevention and control of mildly pathogenic avian influenza (MPAI). Its use has been highly successful in keeping avian influenza (AI) out of commercial poultry worldwide. However, sometimes AI becomes introduced into poultry populations and, when that occurs, biosecurity again is the primary means of controlling the disease. There is agreement that routine serological monitoring, disease reporting, isolation or quarantine of affected flocks, application of strict measures to prevent the contamination of and movement of people and equipment, and changing flock schedules are necessities for controlling AI. There is disagreement as to the disposition of MPAI-infected flocks: some advocate their destruction and others advocate controlled marketing. Sometimes biosecurity is not enough to stop the spread of MPAI. In general, influenza virus requires a dense population of susceptible hosts to maintain itself. When there is a large population of susceptible poultry in an area, use of an inactivated AI vaccine can contribute to AI control by reducing the susceptibility of the population. Does use of inactivated vaccine assist, complicate or interfere with AI control and eradication? Yes, it assists MPAI control (which may reduce the risk of highly pathogenic AI (HPAI)) but, unless steps are taken to prevent it, vaccination may interfere with sero-epidemiology in the case of an HPAI outbreak. Does lack of vaccine assist, complicate or interfere with AI control and eradication? Yes, it assists in identification of sero-positive (convalescent) flocks in a HPAI eradication program, but it interferes with MPAI control (which in turn may increase the risk of emergence of HPAI). A number of hypothetical concerns have been raised about the use of inactivated AI vaccines. Infection of vaccinated flocks, serology complications and spreading of virus by vaccine crews are some of the hypothetical concerns. The discussion of these concerns should take place in a scientific framework and should recognize that control of MPAI reduces the risk of HPAI. That inactivated vaccines have reduced a flock's susceptibility to AI infection, have reduced the quantity of virus shed post-challenge, have reduced transmission and have markedly reduced disease losses, are scientific facts. The current regulations preventing vaccination against H5 or H7 MPAI have had the effect of promoting circulation of MPAI virus in commercial poultry and live poultry markets. In the absence of highly pathogenic avian influenza, there is no justification for forbidding the use of inactivated vaccine.     

Sequence analysis of the neuraminidase genes of avian influenza A viruses isolated from live bird markets in the United States

Neuraminidase (NA) gene of avian influenza viruses isolated from Live Bird Markets (LBMs) on the east coast of the United States was sequenced and analyzed for mutations associated with antiviral resistance. In total, 189 isolates collected from 1994 to 2005 were used in this study. Full length sequences of the NA gene were obtained from 183 of the 189 isolates. Four different lengths of NA gene were observed; 40 isolates had full length (about 1400 nt), 132 isolates had a deletion of 48 nt, 10 isolates had a deletion of 66 nt, and one isolate had a deletion of 72 nt. Amino acid analysis of the sequence data showed point mutations distributed throughout the gene length. None of these deletions was in the catalytic region and most of the mutations were observed in the flanking regions. None of the isolates had mutations which are known to confer antiviral resistance. These results indicate that though NA gene is prone to mutational changes, much of those changes occur outside the catalytic domain.     

Coinfection of avian influenza virus (H9N2 subtype) with infectious bronchitis live vaccine

Avian influenza virus of H9N2 subtype is pathotyped as a non-highly pathogenic virus. However, frequent incidences of avian influenza of high mortality that are caused by H9N2 viruses have been observed in broiler chicken farms in Iran and some other Asian countries. Coinfections or environmental factors may be involved in such cases. Infectious microorganisms have been implicating in taking part in the cases of coinfection. We studied the effect of experimental coinfection of H9N2 avian influenza virus with infectious bronchitis live vaccine, which is used extensively in chicken farms in Iran. Clinical signs, gross lesions, viral shedding and mortality rate of the experimentally infected birds were examined. Coinfection of infectious bronchitis live vaccine and H9N2 avian influenza virus led to an extension of the shedding period of H9N2 virus, increasing the severity of clinical signs and mortality rates, causing macroscopic lesions in the embryos.     

Enhanced immunity to Plasmodium falciparum circumsporozoite protein (PfCSP) by using Salmonella enterica serovar Typhi expressing PfCSP and a PfCSP-encoding DNA vaccine in a heterologous prime-boost strategy

Two Salmonella enterica serovar Typhi strains that express and export a truncated version of Plasmodium falciparum circumsporozoite surface protein (tCSP) fused to Salmonella serovar Typhi cytolysin A (ClyA) were constructed as a first step in the development of a preerythrocytic malaria vaccine. Synthetic codon-optimized genes (t-csp1 and t-csp2), containing immunodominant B- and T-cell epitopes present in native P. falciparum circumsporozoite surface protein (PfCSP), were fused in frame to the carboxyl terminus of the ClyA gene (clyA::t-csp) in genetically stabilized expression plasmids. Expression and export of ClyA-tCSP1 and ClyA-tCSP2 by Salmonella serovar Typhi vaccine strain CVD 908-htrA were demonstrated by immunoblotting of whole-cell lysates and culture supernatants. The immunogenicity of these constructs was evaluated using a "heterologous prime-boost" approach consisting of mucosal priming with Salmonella serovar Typhi expressing ClyA-tCSP1 and ClyA-tCSP2, followed by parenteral boosting with PfCSP DNA vaccines pVR2510 and pVR2571. Mice primed intranasally on days 0 and 28 with CVD 908-htrA(pSEC10tcsp2) and boosted intradermally on day 56 with PfCSP DNA vaccine pVR2571 induced high titers of serum NANP immunoglobulin G (IgG) (predominantly IgG2a); no serological responses to DNA vaccination were observed in the absence of Salmonella serovar Typhi-PfCSP priming. Mice primed with Salmonella serovar Typhi expressing tCSP2 and boosted with PfCSP DNA also developed high frequencies of gamma interferon-secreting cells, which surpassed those produced by PfCSP DNA in the absence of priming. A prime-boost regimen consisting of mucosal delivery of PfCSP exported from a Salmonella-based live-vector vaccine followed by a parenteral PfCSP DNA boosting is a promising strategy for the development of a live-vector-based malaria vaccine.     

H5N1型高致病性禽流感病毒血凝素蛋白及神经氨酸酶的B细胞抗原表位预测

目的预测H5N1亚型高致病性禽流感病毒HA蛋白和NA蛋白的B细胞表位,为基于B细胞表位的预防性疫苗设计提供依据。方法基于HA蛋白和NA蛋白的蛋白质序列,采用Kyte-Doolittle的亲水性方案,Emini方案,Karplus方案和Jameson-wolf抗原指数方案,并辅以MAGE蛋白的二级结构柔性区域分析,预测HA蛋白和NA蛋白的B细胞表位。结果分别预测出了6条血凝素蛋白(Hemagglutinin,HA)以及6条神经氨酸酶(Neuraminidase,NA)B细胞优势表位。结论这些B细胞表位可为禽流感疫苗的研制提供实验依据。     

Virus-like particle (VLP)-based vaccines for pandemic influenza: Performance of a VLP vaccine during the 2009 influenza pandemic

The influenza pandemic of 2009 demonstrated the inability of the established global capacity for egg-based vaccine production technology to provide sufficient vaccine for the population in a timely fashion. Several alternative technologies for developing influenza vaccines have been proposed, among which non-replicating virus-like particles (VLPs) represent an attractive option because of their safety and immunogenic characteristics. VLP vaccines against pandemic influenza have been developed in tobacco plant cells and in Sf9 insect cells infected with baculovirus that expresses protein genes from pandemic influenza strains. These technologies allow rapid and large-scale production of vaccines (3-12 weeks). The 2009 influenza outbreak provided an opportunity for clinical testing of a pandemic influenza VLP vaccine in the midst of the outbreak at its epicenter in Mexico. An influenza A(H1N1)2009 VLP pandemic vaccine (produced in insect cells) was tested in a phase II clinical trial involving 4,563 healthy adults. Results showed that the vaccine is safe and immunogenic despite high preexisting anti-A(H1N1)2009 antibody titers present in the population. The safety and immunogenicity profile presented by this pandemic VLP vaccine during the outbreak in Mexico suggests that VLP technology is a suitable alternative to current influenza vaccine technologies for producing pandemic and seasonal vaccines.     

Determination of the neuraminidase specificity of influenza A and B viruses using a modified lectin test

A highly purified lectin was obtained from peanuts. The lectin test was modified for determination of the neuraminidase activity of influenza viruses. The test was found to be useful for the determination of the antigenic specificity of neuraminidase. It was found to be 10-50 times as sensitive as the Aminoff test and more economic. The potentials of lectin test are discussed.     

Molecular aspects of avian influenza (H5N1) viruses isolated from humans

In 1997, 18 human infections with H5N1 influenza type A were identified in Hong Kong and six of the patients died. There were concomitant outbreaks of H5N1 infections in poultry. The gene segments of the human H5N1 viruses were derived from avian influenza A viruses and not from circulating human influenza A viruses. In 1999 two cases of human infections caused by avian H9N2 virus were also identified in Hong Kong. These events established that avian influenza viruses can infect humans without passage through an intermediate host and without acquiring gene segments from human influenza viruses. The likely origin of the H5N1 viruses has been deduced from molecular analysis of these and other viruses isolated from the region. The gene sequences of the H5N1 viruses were analysed in order to identify the molecular basis for the ability of these avian viruses to infect humans.