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Results demonstrate that the partition (par) region of Agrobacterium tumefaciens plasmid pTAR codes for two proteins instead of one as was previously reported. Data indicate that parA gene codes a protein with a cysteine residue whereas parB coded protein has no cysteine.

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design. IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design. [ABSTRACT FROM AUTHOR]

Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo. [ABSTRACT FROM AUTHOR]

Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo. [ABSTRACT FROM AUTHOR]