PMC

CTL response in HI-infected/667 F(ab′)₂-treated and HI-infected/672 MAb-treated mice. Mice were infected as described in the legend to Fig. . (A) Primary CD8⁺ T-cell response. The presented data are the averages of values obtained from two animals analyzed on day 14 postinfection in each of three independent experiments conducted. (B) Memory CD8⁺ T-cell response. Eight mice per group were used in three independent experiments conducted as indicated in the legend to Fig. . (C) Cytotoxic activity against spleen cells loaded with the GagL peptide in mice subjected to a viral challenge. Experiments were conducted as described for Fig. . Eight mice per group were used in three independent experiments as indicated in the legend to Fig. . Error bars indicate standard deviations.

Design of the experiments. 129/Sv/Ev mice were infected on day 3 after birth with either 5 × 10⁴ (HI-infected mice) or 1 to 5 (LI-infected mice) FrCas^E FFU. HI-infected, but not LI-infected, mice were subjected to immunotherapy with either the 667 MAb (IgG2a/κ), the 667 F(ab′)₂ MAb, or the 672 MAb (IgM/κ) under the conditions indicated in the figure. The 667 F(ab′)₂ MAb was administrated every day with double injections on days 4 and 6 after birth, as indicated by double arrows. Mice were monitored from the day of infection onwards as indicated in the text. The analyzed parameters were (i) serum viremia, (ii) Env-expressing splenocytes, (iii) spleen infectious centers (SICs), (iv) endogenous anti-FrCas^E IgM and IgG responses, and (v) primary and secondary CTL responses against FrCas^E-infected cells. Additional treatments and experiments are indicated in the text.

Characterization of the 672 MAb. (A) Identification of the Env epitope recognized by the 672 MAb. The SPOT analysis was carried out as described in Materials and Methods. Two hundred seventeen overlapping 15-mer amino acid peptides frameshifted by 3 residues and representing the complete CasBr Env were synthesized on a nitrocellulose membrane and probed with 672. Only the region with positive signals is presented. Amino acids are numbered with respect to the first amino acid of the surface protein (pep, peptide; aa, amino acid). (B) Localization of the epitope recognized by 672 in Env. The overall structure of CasBr Env is depicted. The epitope recognized by 672 (gray box) is contained in that recognized by 667 (white box). It is localized within the variable region A (VRA) of the receptor-binding domain (RBD). SS, signal sequence; SU, surface protein; TM, transmembrane protein.

In vivo FcγR-dependent cytolytic activities of the 667 MAb, the 667 F(ab′)₂ fragment, and the 672 MAb. The assessment of FcγR-dependent cytolytic activities of the 667 MAb, the 667 F(ab′)₂ fragment, and the 672 MAb is described in Materials and Methods. Briefly, noninfected and FrCas^E-infected splenocytes differentially labeled with high and low concentrations of the vital dye CSFE were administered intravenously to naive mice or to mice formerly treated with the anti-FcγRII and anti-FcγRIII 2.4G2 MAb. At the same time, the animals received either the 667 MAb, the 667 F(ab′)₂ fragment, or the 672 MAb. Flow cytometry assay of CFSE^low/CFSE^high ratios from cells recovered from spleens 5 h later to allow the comparison of the in vivo cytolytic activity of the anti-FrCas^E MAbs and F(ab′₂) in the presence or in the absence of FcγR-blocking MAb. Two mice per group were used in three independent experiments. Error bars indicate standard deviations.

Humoral responses in HI-infected/immunotherapy-treated and LI-infected/nontreated mice. Anti-FrCas^E IgM (A) and IgG (B) responses developed in HI-infected/667 MAb-treated- and LI-infected/nontreated mice. Forty neonates per group of animals each were used per experiment as described in the legend to Fig. . Two independent experiments were conducted. (C) Anti-FrCas^E neutralization activity in sera from HI-infected/immunotherapy-treated and LI-infected/nontreated mice. Neutralization activity was assayed using sera from 15-week-old mice. The presented data are the averages of values obtained from two groups of 15 mice analyzed in independent experiments. In the left panel, the assays of total neutralization activities in the sera from the various mice were conducted using identical volumes of sera collected from these mice. In the right panel, the assays of specific neutralization activities (neutralization activity per weight unit of anti-FrCas^E immunoglobulin) were conducted after appropriate dilutions of serum samples so as to have the same amounts of anti-FrCas^E immunoglobulins in all samples from HI-infected/immunotherapy-treated mice and from LI-infected/nontreated mice. Anti-FrCas^E IgM (D) and total anti-FrCas^E IgGs (E) responses developed in HI-infected/667 F(ab′)₂-treated and HI-infected/672 MAb-treated mice. Forty neonates per group of animals each were used per experiment as described in the legend to Fig. . Two independent experiments were conducted. Error bars indicate standard deviations.

Viral propagations in HI-infected/immunotherapy-treated mice versus HI-infected/nontreated animals. A total of 54 newborn mice were used per group of HI-infected/667 MAb-treated and HI-infected/nontreated mice (two animals per time point in three different experiments), and a total of 36 newborn mice were used for each of the other groups (two animals per time point in two different experiments). Viral propagation was monitored using three criteria: spleen infectious centers, Env-expressing splenocytes, and serum viremia. (A, D, and F) SIC assays. Spleen cells from simply infected mice or from infected mice treated with either the 667 MAb, the 667 F(ab′)₂ fragment, or the 672 MAb were isolated and plated onto indicator Mus dunni cells. SICs corresponded to the number of infectious centers scored in focal immunofluorescence assays after 3 days of coculture, as described in Materials and Methods. % SICs on the y axis indicates the percentage of infectious splenocytes versus the total number of splenocytes used in the experiment. (B, E, and G) Assay of Env-expressing splenocytes. Splenocytes were purified from the various mice, and Env expression at their surface was quantified by flow cytometry, as described in Materials and Methods. The percentage of Env-expressing splenocytes is indicated on the y axis. (C and H) Serum viremia assay. Viremia was assayed in a focal immunofluorescence assay, as indicated in Materials and Methods. Error bars indicate standard deviations. Statistical significance between the groups was established using the Mann and Whitney test. **, P < 0.01; *, P < 0.05.

CTL response in HI-infected/667 MAb-treated and LI-infected/nontreated mice. Mice were infected as described in Fig. using age-matched noninfected/nontreated mice as controls. (A) Primary CD8⁺ T-cell response. Typical flow cytometry analyses of D^b-GagL⁺ CD8⁺ T cells performed on day 14 postinfection are presented. (B) Kinetic analysis of the primary CD8⁺ T-cell response. The presented data are the averages of values obtained from two mice per time point in each of three independent experiments conducted. (C) Memory CD8⁺ T-cell response. Typical flow cytometry analysis of D^b-GagL⁺ CD8⁺ T cells carried out 10 days after a viral challenge performed 8 weeks postinfection. (D) Statistical analysis of the memory CD8⁺ T-cell response. Eight mice per group were used in three independent experiments conducted as indicated in panel C. (E) Cytotoxic activity against spleen cells loaded with the GagL peptide in mice subjected to a viral challenge. Splenocytes were loaded with GagL or a control peptide and stained in the presence of low and high concentrations of CSFE, respectively. They were administered to mice 10 days after viral challenge in week 8 postinfection. Flow cytometry analyses were carried out on the following day. Typical data are presented in the figure. The left panel indicates the proportion of control- and GagL peptide-loaded splenocytes administered to mice. A reduction in CFSE^High staining (right peak) in the other two panels is indicative of infected cell lysis. (F) Statistical analysis of cytotoxic activity against spleen cells loaded with the GagL peptide in mice subjected to a viral challenge. Eight mice per group were used in three independent experiments as indicated in panel E. Error bars indicate standard deviations.