Live-Attenuated Respiratory Syncytial Virus Vaccine Candidate With Deletion of RNA Synthesis Regulatory Protein M2-2 is Highly Immunogenic in Children

Abstract

Background: Live respiratory syncytial virus (RSV) candidate vaccine LIDΔM2-2 is attenuated by deletion of the RSV RNA regulatory protein M2-2, resulting in upregulated viral gene transcription and antigen expression but reduced RNA replication.

Methods: RSV-seronegative children ages 6-24 months received a single intranasal dose of 105 plaque forming units (PFU) of LIDΔM2-2 (n = 20) or placebo (n = 9) (NCT02237209, NCT02040831). RSV serum antibodies, vaccine infectivity, and reactogenicity were assessed. During the following RSV season, participants were monitored for respiratory illness and pre- and post-RSV season serum antibodies.

Results: Vaccine virus was shed by 95% of vaccinees (median peak titers of 3.8 log10 PFU/mL by quantitative culture and 6.3 log10 copies/mL by PCR); 90% had ≥4-fold rise in serum neutralizing antibodies. Respiratory symptoms and fever were common in vaccine (95%) and placebo (78%). One vaccinee had grade 2 rhonchi concurrent with vaccine shedding, rhinovirus, and enterovirus. Eight of 19 vaccinees versus 2 of 9 placebo recipients had substantially increased RSV antibody titers after the RSV season without medically attended RSV disease, indicating anamnestic vaccine responses to wild-type RSV without significant illness.

Conclusion: LIDΔM2-2 had excellent infectivity and immunogenicity, encouraging further study of vaccine candidates attenuated by M2-2 deletion.

Clinical trials registration: NCT02237209, NCT02040831.

Figure 1.

Serum respiratory syncytial virus (RSV) antibody titers in vaccine and placebo recipients. Serum RSV 60% plaque reduction neutralizing titers (PRNT₆₀) (A) and anti-RSV F IgG titers (B) were determined by complement-enhanced 60% plaque reduction neutralization assay and IgG-specific enzyme-linked immunosorbent assay (ELISA) against purified RSV F protein, respectively, for vaccine (open circles) and placebo (×) recipients in sera collected at preinoculation (screening), postinoculation (study day 56), and postsurveillance (after the RSV season, 1 to 30 April in the calendar year following the inoculation). Titers are expressed as the reciprocal log₂. The lines indicate median (solid line) and mean value (dashed line). P values were determined by Wilcoxon rank sum test. One vaccine recipient who did not shed vaccine virus is indicated with an asterisk; this infant was lost to follow-up at the postsurveillance visit and the data for this time point are missing.

Figure 2.

Reverse cumulative distribution of serum respiratory syncytial virus (RSV) neutralizing antibody titer postinoculation. Serum RSV plaque reduction neutralizing titers (PRNT₆₀) determined by complement-enhanced 60% plaque reduction neutralization assay in sera collected at 56 days after inoculation are expressed as reciprocal log₂ for vaccine (solid line) and placebo (dashed line) recipients. For ease of interpretation, the arithmetic value (1:64) at the titer achieved by 85% of vaccinees is indicated.

Figure 3.

Correlation of the peak titer of vaccine virus in nasal washes and serum respiratory syncytial virus (RSV) antibody titer. Correlation of peak vaccine virus titers shed in nasal wash (NW), determined by culture (A, C) and RT-qPCR (B, D) with the change from baseline to study day 56 in anti-RSV F IgG titer (A, B) and RSV serum neutralizing antibody titer (C, D). Titers of vaccine virus determined by immunoplaque assay and expressed as log₁₀ transformed number of plaque-forming units (PFU) per mL of NW. Log₁₀ copy numbers of vaccine virus were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) specific for the RSV M gene. The limit of virus detection by titration and by RT-qPCR was 0.5 log₁₀ PFU/mL and 1.7 log₁₀ copy numbers/mL; samples below the limit of detection for the plaque assays and RT-qPRC were assigned a titer of 0.5 log₁₀ PFU/mL and 1.7 log₁₀ copy numbers/mL, respectively. Serum RSV 60% plaque reduction neutralizing titers (PRNT₆₀) and anti-RSV F IgG titers were determined by complement-enhanced 60% plaque reduction neutralization assay and IgG-specific enzyme-linked immunosorbent assay (ELISA) against purified RSV F protein, respectively, and titers expressed as reciprocal log₂. Samples below the limit of detection were assigned the reciprocal titers of 2.3 log₂ (PRNT₆₀) and 4.6 log₂ (ELISA). Spearman correlation coefficients and P values are shown. The regression lines are included to give a descriptive summary of the directionality of the association, but statistical analysis consists of Spearman correlations, which avoid the assumption of normality.

Figure 4.

Rises in serum respiratory syncytial virus (RSV) neutralizing antibody titers, and incidences of medically attended RSV illness, during the RSV season surveillance. Serum RSV plaque reduction neutralizing titers (PRNT₆₀) in sera collected pre- and post-RSV season surveillance shown for vaccine (left) and placebo (right) recipients who had a 4-fold or greater increase in either serum RSV PRNT₆₀ or anti-RSV F IgG titer. Dashed and solid lines indicate subjects with and without a medically attended RSV-associated illness during surveillance. Note, 2 participants with ≥4-fold increase in anti-RSV F IgG titer had a smaller increase in serum neutralizing antibody titer. Titers are expressed as the reciprocal log₂, but for ease of interpretation, titers corresponding to the arithmetic values of 1:20, 1:131, 1:500, 1:1333, 1:2988, and 1:13507 are indicated.