Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease.

Respiratory syncytial virus (RSV) is a leading cause of hospitalization in infants. A formalin-inactivated RSV vaccine was used to immunize children and elicited nonprotective, pathogenic antibody. Immunized infants experienced increased morbidity after subsequent RSV exposure. No vaccine has been licensed since that time. A widely accepted hypothesis attributed the vaccine failure to formalin disruption of protective antigens. Here we show that the lack of protection was not due to alterations caused by formalin but instead to low antibody avidity for protective epitopes. Lack of antibody affinity maturation followed poor Toll-like receptor (TLR) stimulation. This study explains why the inactivated RSV vaccine did not protect the children and consequently led to severe disease, hampering vaccine development for 42 years. It also suggests that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR agonists in their formulation, and it identifies affinity maturation as a key factor for the safe immunization of infants.

Natural history of human respiratory syncytial virus inferred from phylogenetic analysis of the attachment (G) glycoprotein with a 60-nucleotide duplication.

A total of 47 clinical samples were identified during an active surveillance program of respiratory infections in Buenos Aires (BA) (1999 to 2004) that contained sequences of human respiratory syncytial virus (HRSV) with a 60-nucleotide duplication in the attachment (G) protein gene. This duplication was analogous to that previously described for other three viruses also isolated in Buenos Aires in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003). Phylogenetic analysis indicated that BA sequences with that duplication shared a common ancestor (dated about 1998) with other HRSV G sequences reported worldwide after 1999. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but both copies of the duplicated segment accumulated nucleotide substitutions in more recent viruses at a rate apparently higher than in other regions of the G protein gene. The evolution of the viruses with the duplicated G segment apparently followed the overall evolutionary pattern previously described for HRSV, and this genotype has replaced other prevailing antigenic group B genotypes in Buenos Aires and other places. Thus, the duplicated segment represents a natural tag that can be used to track the dissemination and evolution of HRSV in an unprecedented setting. We have taken advantage of this situation to reexamine the molecular epidemiology of HRSV and to explore the natural history of this important human pathogen.

Genetic and antigenic variability of human respiratory syncytial virus (groups a and b) isolated over seven consecutive seasons in Argentina (1995 to 2001).

The genetic and antigenic variability of human respiratory syncytial virus (HRSV) strains isolated in Buenos Aires from 1995 to 2001 was evaluated by partial nucleotide sequencing of the G gene and enzyme-linked immunosorbent assay analysis with anti-G monoclonal antibodies. Phylogenetic analyses showed that 37 group A strains clustered into five genotypes, whereas 20 group B strains clustered into three genotypes. Group A showed more genetic variability than group B. A close correlation between genotypes and antigenic patterns was observed. Changes detected in the G protein of viruses from both groups included (i) amino acid substitutions and(ii) differences in protein length due to either changes in stop codon usage or sequence duplications. Three B strains from 1999 exhibited a duplication of 20 amino acids, while one B strain from 2001 had 2 amino acids duplicated. The comparison among Argentinean HRSV strains and viruses isolated in other geographical areas during different epidemics is discussed.

Diferentes patrones de circulación dentro de los subgrupos A y B del virus sincitial respiratorio humano en algunas provincias de Cuba / Different patterns of circulation in subgroups A and B of human respiratory syncytial virus in some provinces of Cuba

Se realizó la secuenciación nucleotídica de la región del tercio C-terminal de la proteína G de 37 muestras de exudados nasofaríngeos, de niños menores de 1 año provenientes de algunas provincias de Cuba durante 5 períodos epidémicos (1995-2000), para conocer los patrones de circulación de cepas del virus sincitial respiratorio humano; el cual se clasifica en 2 subgrupos antigénicos A y B, y cada uno contiene múltiples variantes. El subgrupo A circuló durante todos los años, el subgrupo B se detectó solamente durante el año 2000. Dentro del subgrupo A se observó la presencia de cepas con 2 tamaños diferentes de la proteína G (297 aa y 298 aa), mientras que para el subgrupo B fue observado un único tamaño (295 aa). El análisis filogenético permitió identificar 5 y 2 genotipos dentro de los subgrupos A y B, respectivamente. Los virus de Cuba se relacionaron filogenéticamente con cepas de otras partes del mundo. Dentro del subgrupo A se encontraron 2 cepas, las cuales fueron muy similares a la cepa prototipo Long. Casi todas las cepas del año 2000 de ambos subgrupos, se agruparon filogenéticamente con cepas que circularon en Sudáfrica durante ese mismo período

[Different circulation patterns of subgroups A and B of human respiratory syncytial virus in some provinces of Cuba]. / Diferentes patrones de circulación dentro de los subgrupos A y B del virus sincitial respiratorio humano en algunas provincias de Cuba.

The nucleotide sequencing of the protein G C-terminal region of 37 samples taken from nasopharyngeal washings of under one-year old children from some Cuban provinces was made for 5 epidemic periods (1995-2000) to find out the circulation patterns of strains of human respiratory syncytial virus that is classified in two antigenic subgroups known as A and B; each of them contains multiple variants. Subgroup A has circulated during all these years but subgroup B was detected only in the year 2000. The presence of strains with two different sizes of protein G (297 aa and 298 aa) was observed whereas subgroup B showed only one size (295 aa). Phylogenetic analysis allowed identifying 5 and 2 genotypes within subgroups A and B respectively. Viruses present in Cuba were phylogenetically related to the strains of other parts of the world. Subgroup A comprised two strains very similar to Long prototype strain. Almost all the strains of both subgroups in the year 2000 phylogenetically related with strains that circulated in South Africa during the same period.

Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides.

The entire nucleotide sequence of the G gene of three human respiratory syncytial virus (HRSV) isolates (antigenic group B) has been determined. These three viruses (named BA viruses) were isolated in Buenos Aires in 1999 from specimens collected in different hospitals and at different dates. BA viruses have an exact duplication of 60 nucleotides in the G gene, starting after residue 791. This duplication is flanked by a repeat of four nucleotides (GUGU) and can fold into a relatively stable secondary structure. These features suggest a possible mechanism for the generation of a duplicated G segment. The predicted polypeptide is lengthened by 20 amino acids (residues 260-279) and this is reflected in the slower electrophoretic mobility of the G protein precursor of BA viruses compared with related viruses. The changes reported here expand the examples of drastic genetic alterations that can be introduced into the G protein sequence of HRSV while it replicates in its natural host.

Antigenic and Genetic Characterization of Twenty-six Strains of Human Respiratory Syncytial Virus (Subgroup A) Isolated During Three Consecutive Outbreaks in Havana City, Cuba

Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100 percent) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus