Protective antibodies against human parainfluenza virus type 3 infection

Abstract

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two nonoverlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals.

Methods to Measure Antibody Neutralization of Live Human Coronavirus OC43

“…For individual B cells sorted onto feeder cells, supernatant was removed after 13 days of culture, plates were immediately frozen on dry ice, stored at −80 • C, thawed, and RNA was extracted using the RNeasy Micro Kit (Qiagen, Germantown, MD, cat#74034). The entire eluate from the RNA extraction was used in the reverse transcription and two rounds of PCR, as previously described [26]. Sequences were analyzed using IMGT/V-Quest to identify V, D, and J gene segments.…”

“…Sequences were analyzed using IMGT/V-Quest to identify V, D, and J gene segments. Paired heavy chain VDJ and light chain VJ sequences were cloned into pTT3-derived expression vectors containing the human IgG1, Igκ, or Igλ constant regions using In-Fusion cloning (Clontech, Mountain View, CA, USA, cat#638911) as previously described [26,27].…”

“…We then enriched for OC43 S2P-binding B cells from the spleen of a human donor using magnetic microbeads conjugated to antibodies targeting PE (Figure 2a). We included a "decoy" tetramer consisting of biotinylated HPIV1 postF and streptavidin conjugated to PE/DyLight650 to allow for identification and exclusion of B cells binding non-specifically and B cells binding to PE or streptavidin [26,[29][30][31][32]. We sorted individual isotype-switched, decoy negative, OC43 S2P-binding B cells onto irradiated 3T3 feeder cells expressing CD40L, IL-2, and IL-21 in 96-well plates, as described [19,26].…”

“…We included a "decoy" tetramer consisting of biotinylated HPIV1 postF and streptavidin conjugated to PE/DyLight650 to allow for identification and exclusion of B cells binding non-specifically and B cells binding to PE or streptavidin [26,[29][30][31][32]. We sorted individual isotype-switched, decoy negative, OC43 S2P-binding B cells onto irradiated 3T3 feeder cells expressing CD40L, IL-2, and IL-21 in 96-well plates, as described [19,26]. We screened culture supernatants qualitatively for neutralization of OC43 by mixing 40 µL of supernatant with 50 TCID 50 of OC43 and assessing each well for CPE on day 8 postinfection.…”

“…OC2 did not bind to the spike proteins of other coronaviruses (Figure 2d). jugated to PE/DyLight650 to allow for identification and exclusion of B cells binding nonspecifically and B cells binding to PE or streptavidin [26,[29][30][31][32]. We sorted individual isotype-switched, decoy negative, OC43 S2P-binding B cells onto irradiated 3T3 feeder cells expressing CD40L, IL-2, and IL-21 in 96-well plates, as described [19,26].…”