Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro : Clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

Abstract

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52͞ 2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription-PCRamplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.Respiratory syncytial virus (RSV), the leading cause of severe viral respiratory illness in pediatric populations throughout the world (reviewed in ref. 1), accounts for approximately 90,000 hospitalizations in infants and children in the United States each year (2). The importance of RSV as a respiratory pathogen makes development of a safe and effective RSV vaccine a public health priority (3). Although a number of approaches to RSV vaccine development have been taken, live RSV vaccines may provide the best alternative for immunizing young infants, because a live vaccine would mimic natural infection, induce a balanced cellular and humoral immune response, and be unlikely to produce enhanced disease (4).RSV exists as two antigenically distinct subgroups, A and B, and both RSV A and RSV B infections are capable of inducing severe lower respiratory tract disease (5-7). For this reason, a bivalent live RSV vaccine containing attenuated RSV A and RSV B components would be most desirable. Recently, a live attenuated RSV A candidate vaccine has been identified that appears to be safe and immunogenic in infants and children over 6 months of age (8). In addition, a cold-passaged (cp) RSV B candidate vaccine, designated RSV B1 cp-52͞2B5 (cp-52), was derived by passage of the RSV B1 wild-type (wt) virus 52 times at low temperature (21-32°C) (9). Cp-52 was shown to be restricted in replication in vivo but still able to induce RSV serum-neutralizing antibody responses in cotton rats, African green monkeys, and chimpanzees (9). Also, it was found to be phenotypically stable after prolonged replication in cotton rats (9). Here, we describe the phase I evaluation of the cp-52 candidate vaccine in adults, children, and infants. Although this virus mutant grew to high titer (Ͼ10 7....

Subgroup C avian metapneumovirus (MPV) and the recently isolated human MPV exhibit a common organization but have extensive sequence divergence in their putative SH and G genes

“…Between HRSV subgroups A and B, the SH protein is mostly conserved (71?9 % aa identity between representatives of the two subgroups; Chen et al, 2000) and the short HRSV SH protein (64 and 65 aa in subgroups A and B, respectively) is suspected to have its integrity maintained due to an evolutionary or biological pressure (Chen et al, 2000). Experiments with HRSV and BRSV mutants or recombinant viruses deleted in their SH gene (Karron et al, 1997;Bukreyev et al, 1997;Techaarpornkul et al, 2001;Karger et al, 2001) showed that SH is dispensable for virus growth in vitro but that a lack of SH may, in a murine experimental model, selectively impair HRSV replication in the upper respiratory tract and not in the lung (Bukreyev et al, 1997). Whether a lower biological pressure in APV explains a greater variation among the SH genes of different APV subgroups or whether different SH proteins have a critical role in the host range, pathogenesis or organ tropism of MPVs awaits further investigation.…”

Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein

“…The smaller SH protein is considered to act like a viroporin and increases membrane permeability (5). Of these envelope glycoproteins, only the RSV F protein is indispensable for viral replication in vitro (9). It is the most conserved RSV glycoprotein and also the main target of neutralizing antibodies and vaccine development (10,11).…”

Lactoferrin and surfactant protein A exhibit distinct binding specificity to F protein and differently modulate respiratory syncytial virus infection

“…A deletion mutant of RSV lacking both G and SH genes was able to replicate efficiency in certain cell lines and to produce syncytia [23]; and F protein by itself could induce proinflammatory cytokines in human monocytes [24]. Therefore, it is considered that the interaction of SP-A or LF with F protein must be the important mechanism by which SP-A and LF modulate the RSV infection to HEp-2 cells.…”