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Eb Avg secondary scorec Group mortalityI IIMock (PBS) gD gHt-gL Mock (PBS) gD gHt-gL10.5 5.5 2.8 22.3 5.8 4.16.5 0 0 19.6 05/10 0/10 0/9 5/5 0/5 0/FIG. 6. Blocking of HSV entry by rabbit antibodies to gHt-gL. (A) HSV1(hrR3) was incubated for 90 min at 37 with various concentrations of rabbit anti-gHt-gL serum R136, R137, R138, or R139, and the serum-virus mixture was added to Vero cell monolayers
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L monolayers at 4 for 90 min. The medium was removed, various dilutions of either R83, R137, or LP11 were added, and monolayers were incubated at 4 for 90 min and then at 37 for 5 h. Virus entry was assayed as described for panel A.a In experiment I, there were 10 mice in each group. One mouse in the gH-gL-immunized group of experiment I died prior to challenge. In experiment II, there were fiv
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Days 3 to 8. Score range was 0 to 10.molecular-weight forms (Fig. 5B). The composition of these bands remains to be determined. All four sera exhibited significant titers of complement-independent HSV-1 neutralizing activity (Table 1). In addition, these sera also neutralized HSV-2, albeit at a much reduced potency. These results indicated that the immunizing protein had biologic activity. In addi
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Rus adsorption (Fig. 6B). R137 had blocking activity similar to that observed for LP11, indicating that both antibodies recognized a site on gH-gL which was critical for postbinding steps in virus entry. This experiment suggests that the gHt-gL complex used to prepare R137 contains a functionally active conformation. In contrast, antibody R83 was unable to block virus entry. This was an important
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Cording to experiment I (Table 2). The serum-virus mixture was added to Vero cell monolayers in a 96-well plate, incubated at 4 for 90 min, and then incubated at 37 for 5 h. Virus entry was assayed as an increase in -galactosidase activity in cytoplasmic extracts from each well and expressed as percentages of control values obtained with virus alone. (B) Same as panel A except that the sera were
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Th HSV-1(hrR3) (22) and measuring -galactosidase activity at 5 h postinfection. The neutralization titer represents the dilution of antiserum needed to reduce -galactosidase activity to 50 of the maximum seen with no serum added. b Virus infection was measured by plaque formation by HSV-1(KOS) on Vero cells, using the black plaque assay. The neutralization titer represents the dilution of antiser
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Mals produced antibodies which recognized gHt and gL on a Western blot of a denaturing gel (Fig. 5A). On a Western blot of a nondenaturing (native) gel (6), these antibodies also recognized higherTABLE 1. HSV neutralizing activity of sera from rabbits immunized with gHt-gLVirus neutralization titer (50 ) Adjuvant Rabbit Entry assay,a HSV-1(hrR3) Plaque assayb HSV-1(KOS) HSV-2(333)Freund's R136 R13
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Eb Avg secondary scorec Group mortalityI IIMock (PBS) gD gHt-gL Mock (PBS) gD gHt-gL10.5 5.5 2.8 22.3 5.8 4.16.5 0 0 19.6 05/10 0/10 0/9 5/5 0/5 0/FIG. 6. Blocking of HSV entry by rabbit antibodies to gHt-gL. (A) HSV1(hrR3) was incubated for 90 min at 37 with various concentrations of rabbit anti-gHt-gL serum R136, R137, R138, or R139, and the serum-virus mixture was added to Vero cell monolayers