Characterization of Gene Use and Efficacy of Mouse Monoclonal Antibodies toStreptococcus pneumoniaeSerotype 8

Abstract

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugatebased vaccines, the prevalence of antibiotic-resistant pneumococcal strains, serotype (ST) replacement in nonconjugate vaccine strains, and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study, we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs, including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC, were produced, and their nucleotide sequences, epitope and fine specificity, and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V H ) and light (V L ) chain genes, with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V H gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy.

The innate immune response to Streptococcus pneumoniae in the lung depends on serotype and host response

“…The propensity of ST8 to invade the bloodstream in mice mirrors clinical data from a recent study showing that the most common ST causing invasive pneumococcal disease was ST8 [23]. The invasiveness of 6308 in mice [12;26] and ST8 strains in humans could be an effect of the non-hemolytic pneumolysin expressed by 6308 and other ST8 strains [12;33]. Hemolytic pneumolysin was required for a protective host response to pneumococcus in mice [33;16], but its absence increased lethality of A66.1 in one model [16].…”

“…To induce pneumococcal pneumonia, mice were anaesthetized with isofluorane and intranasally (i.n.) inoculated as described previously [11;13;16;26]. To determine the amount of bacteria actually administered, the inoculum was cultured on trypticase soy agar plates with 5% sheep’s blood (Becton Dickinson, Sparks, MD) before and after infection of mice.…”

“…Mice infected with the aforementioned inocula (5×10 5 CFU for A66.1 and 5×10 3 CFU for 6308) were killed 24 hours post-infection and lung tissue was removed aseptically, washed in 5ml of sterile PBS and homogenized in 6ml of Trizol (Invitrogen, Carlsbad, CA) as described [26]. Total RNA was extracted using Trizol according to manufacturer’s instructions and purified with RNeasy kit (Qiagen, Valencia, CA).…”

Reduction of Streptococcus pneumoniae Colonization and Dissemination by a Nonopsonic Capsular Polysaccharide Antibody

“…The derivation and biological activities of PPS3 mouse IgG1 MAbs, 1E2 and 7A9, and the PPS8 mouse IgG1 MAb, 31B12, which was used as a specificity and isotype control, were previously described ( 19 , 50 ). MAb F(ab′) 2 fragments were generated with the Mouse IgG1 Fab and F(ab′) 2 Micro Preparation kit (Thermo) according to the manufacturer’s instructions.…”

A Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibody Requires Fcγ Receptor III and Macrophages To Mediate Protection against Pneumococcal Pneumonia in Mice

“…None of the MAbs required complement to mediate protection in Wt mice (49). An IgG1 MAb that binds PPS8, 31B12 (56), was used as an isotype control.…”