Human monoclonal antibodies broadly neutralizing against influenza B virus.

Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus.

Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses.

Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses.

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Development and evaluation of an immunochromatographic assay to detect serum anti-leptospiral lipopolysaccharide IgM in acute leptospirosis.

Leptospirosis is a common life-threatening disease worldwide. However, its diagnosis is frequently ineffective because the gold standard bacterial culture and microscopic agglutination test (MAT) are usually positive 1-2 weeks after the disease onset. We thus developed an immunochromatographic assay (LEPkit) to detect serum anti-leptospiral lipopolysaccharide (LPS) IgM for rapid diagnosis of acute leptospirosis. Using referenced sera of 77 leptospirosis and 91 non-leptospirosis cases, LEPkit yielded 97.4% sensitivity, 94.5% specificity, 93.8 positive predictive value (PPV), 97.7% negative predictive value (NPV), and 95.8% accuracy. The stability of this kit stored for up to 18 months and its reproducibility were confirmed. Testing in 74 new cases using samples at admission-phase and subsequent paired samples (total n = 135), overall sensitivity was 98.5%, whereas that of culture and single MAT (≥1:400) was 15.6% and 35.6%, respectively. When only the samples at admission-phase were used (n = 74), the sensitivity remained at 98.7%, whereas that of culture and single MAT (≥1:400) was 28.4% and 13.5%, respectively. In summary, our LEPkit was far more effective than any conventional methods for the diagnosis of acute leptospirosis, especially within the first few days after the disease onset. The ease of use, stability and reproducibility further enhance its feasibility for clinical use on-site.

Emerging antigenic variants at the antigenic site Sb in pandemic A(H1N1)2009 influenza virus in Japan detected by a human monoclonal antibody.

The swine-origin pandemic A(H1N1)2009 virus, A(H1N1)pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1)pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E) in the hemagglutinin (HA) protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1)pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1)pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently.

The impact of antigenic drift of influenza A virus on human herd immunity: Sero-epidemiological study of H1N1 in healthy Thai population in 2009.

To examine the effect of the antigenic drift of H1N1 influenza viruses on herd immunity, neutralization antibodies from 744 sera from Thai healthy volunteers in 2008-2009, who had not been vaccinated for at least the last 5 years, were investigated by microneutralization (MN) and hemagglutination inhibition (HI) assays. Significantly higher MN titers were observed for the H1N1 Thai isolate in 2006 than in 2008. The results indicate that the antigenically drifted virus effectively escaped herd immunity. Since the low neutralization activity of herd immunity against drifted viruses is an important factor for viruses to spread efficiently, continuous sero-epidemiological study is required for public health.

Genotypic characterization of HIV type 1 env gp160 sequences from three regions in Thailand.

We report 25 env gp160 sequences from patients in three geographically distinct districts of Thailand, i.e., Lampang in the north, Trang in the south and Rayong in the east. One of these is a CRF01_AE/subtype B recombinant and the other 24 sequences are purely CRF01_AE. Very little interpopulation diversity was observed between the sequences from the three different geographic regions and from those previously reported by our laboratory from central Thailand. Potential N-linked glycosylation sites (PNLGs) were reasonably conserved among the 25 sequences: we found 15 highly conserved PNLGs on gp120 and 4 almost fully conserved PNLGs on gp41. Analysis of coreceptor tropism revealed that six of the isolates were dual tropic and the others were R5 tropic. We also examined a rare seven amino acid deletion found in one isolate at position 847-853 on gp41. These results may enhance our understanding of HIV-1 currently circulating in Thailand.