Antibody response against other antigens. The neutralizing antibody response after infection with 2 × 10⁶ pfu of VSV serotype New Jersey was determined for TgH(KL25), TgH(VI10), and WT mice (Upper). The anti-keyhole limpet hemocyanin (KLH) antibody response was monitored after i.p. immunizations with 100 μgofKLH in alum on days 0 and 7 (Lower). The data shown represent the mean ± SD of at least two values.

Analysis of the virus-neutralizing antibody response in TgH(KL25) and TgH(VI10) mice. (A) Total Ig-neutralizing titers were determined in serum from naïve C57BL/6, TgH(KL25), and TgH(VI10) mice. The detection limit of the assay is indicated by a dashed line. Note the different scales for LCMV and VSV. (B) C57BL/6 and TgH(KL25) mice were infected i.v. with 2 × 10⁶ pfu of LCMV-WE, serum samples were collected at the time points indicated, and total Ig- and IgG-neutralizing titers were determined. The IgG detection limit of the assay is indicated by a dashed line. (C) C57BL/6 and TgH(VI10) mice were infected i.v. with 2 × 10⁶ pfu of VSV-IND. Total Ig- and IgG-neutralizing titers were determined in the serum. (D)(Left) CD4-depleted TgH(KL25) mice were infected with 2 × 10⁶ pfu of LCMV-WE, and virus-neutralizing antibodies were determined from 10-fold prediluted sera. (Right) Neutralizing antibody response elicited in CD4-depleted TgH(VI10) mice after infection with 2 × 10⁶ pfu of VSV. Sera were 40-fold prediluted. In A, data represent the mean ± SD of five mice from one experiment. The data shown in B and C display the mean ± SD of three mice and represent one of three similar experiments. Data shown in D represent the mean ± SD of at least two animals with one of two experiments shown.

Impact of preexisting or early inducible neutralizing antibodies on virus dissemination and liver pathology. (A) Clearance of LCMV-WE from spleen, kidney, liver, and blood of TgH(KL25) and C57BL/6 mice after i.v. infection with a high (2 × 10⁶ pfu; HD) or a low (200 pfu; LD) dose of LCMV-WE. The detection limit for each organ is indicated by a dashed line, and * indicates data points where a filled triangle is covered by a filled box. Data represent mean ± SD of three animals with one of two experiments shown. (B) Viral titers in spleen, kidney, liver, lung, brain, and blood of TgH(VI10) and C57BL/6 mice determined 2 h after infection with 10⁹ pfu of VSV-IND i.v. Data represent the mean ± SD of at least five animals with one of two experiments shown. (C) Comparison of the liver pathology in KL25 transgenic and WT mice. Activity of ALT and AST was determined in the serum of mice infected with 2 × 10⁶ pfu of LCMV-WE. Data represent the mean ± SD of three animals with one of two experiments shown.

Idiotypic and histological analysis of TgH(KL25) and TgH(VI10) mice. (A) Expression of the KL25 heavy chain in TgH(KL25) mice. Peripheral blood lymphocytes from TgH(KL25) and C57BL/6 mice were stained with anti-B220 in combination with either IIIC4.8 (anti-KL25H) or B2.5 (anti-KL25HL). The percentage of B cells expressing the transgenic heavy chain or heavy/light chain combination is indicated in the corresponding quadrant. Data shown are representative of at least three experiments. (B) Analysis of hybridoma prepared from naïve and LCMV-infected TgH(KL25) mice. Spleen cells from naïve and day 4-infected mice (2 × 10⁶ pfu of LCMV-WE i.v.) were fused to the myeloma X63Ag8.653 and subjected to hypoxanthine/aminopterin/thymidine selection. Surviving clones were expanded, and supernatants were analyzed by ELISA for the presence of the KL25 heavy chain (IIIC4.8) or the KL25 heavy/light chain combination (B2.5). In addition, supernatants were analyzed for LCMV-WE neutralizing activity and the presence of infectious virus. Supernatants referred to as double negative did not bind to either of the anti-KL25 mAbs and might also include Ig-nonproducing clones. (C) Localization of LCMV-specific B cells in spleen during infection with 2 × 10⁶ pfu of LCMV-WE. Cryosections were stained with anti-KL25H-FITC (IIIC4.8, green) and anti-KL25HL-tetramethylrhodamine isothiocyanate (B2.5, red). Digital photos were taken and electronically overlaid. (D) Expression of the VI10 heavy chain in TgH(VI10) mice. Because no mAb was available directed against the heavy chain of VI10, the expression level of the VI10 heavy chain was assessed indirectly with mAb 35.61 specific for the VI10 heavy and light chain combination. This was achieved by breeding TgH(VI10) mice to the VI10-light chain expressing transgenic mouse strain YEN. Peripheral blood lymphocytes from double transgenic, the parental single transgenics, and WT mice were stained with mAb 35.61 in combination with anti-B220 and analyzed by flow cytometry. The percentage of B cells expressing heavy and light VI10-Ig chains are indicated. Data shown are representative of at least three experiments. (E) Splenic localization of VSV-specific B cells. C57BL/6 and TgH(VI10) mice were immunized with 2 × 10⁶ pfu of VSV-IND. Spleen sections from infected (d20) and naïve mice were stained for VSV-specific B cells (red) as described in Materials and Methods. (F) Localization of VSV antigen 2 h after 10⁹ pfu of VSV-IND i.v. infection. Spleen sections were stained for VSV (red) as described in Materials and Methods.