Live Attenuated Influenza Vaccine contains Substantial and Unexpected Amounts of Defective Viral Genomic RNA

Abstract

The live attenuated influenza vaccine FluMist^® was withdrawn in the USA by the Centers for Disease Control and Prevention after its failure to provide adequate protective immunity during 2013-2016. The vaccine uses attenuated core type A and type B viruses, reconfigured each year to express the two major surface antigens of the currently circulating viruses. Here Fluenz™ Tetra, the European version of this vaccine, was examined directly for defective-interfering (DI) viral RNAs. DI RNAs are deleted versions of the infectious virus genome, and have powerful biological properties including attenuation of infection, reduction of infectious virus yield, and stimulation of some immune responses. Reverse transcription polymerase chain reaction followed by cloning and sequencing showed that Fluenz™ vaccine contains unexpected and substantial amounts of DI RNA arising from both its influenza A and influenza B components, with 87 different DI RNA sequences identified. Flu A DI RNAs from segment 3 replaced the majority of the genomic full-length segment 3, thus compromising its infectivity. DI RNAs arise during vaccine production and non-infectious DI virus replaces infectious virus pro rata so that fewer doses of the vaccine can be made. Instead the vaccine carries a large amount of non-infectious but biologically active DI virus. The presence of DI RNAs could significantly reduce the multiplication in the respiratory tract of the vaccine leading to reduced immunizing efficacy and could also stimulate the host antiviral responses, further depressing vaccine multiplication. The role of DI viruses in the performance of this and other vaccines requires further investigation.

Keywords: defective interfering RNA; influenza A; influenza B; live attenuated vaccine.

Figure 1

(A) Diagram of the general genetic organisation of influenza virus defective-interfering (DI) RNAs. The central deletion between positions labelled x and y is highly variable in length but the 5′ and 3′ termini (solid) are retained. (B)Representative analysis of the products of reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific for each of segments 1, 2, and 3 of the influenza A and influenza B strains present in the Fluenz™ Tetra vaccine (batch CH2020). Products representing full-length influenza segments are indicated by arrows; boxed areas indicate the regions excised for gel extraction for putative DI RNAs. Influenza A segment 1 (lanes 1–2), segment 2 (lanes 3–4), segment 3 (lanes 5–8). Influenza B segment 1 (lanes 9–10), segment 2 (lanes 11–12), segment 3: (lanes 13–14). Products were amplified for 30 cycles except for lanes 7–8 which required 35 cycles to visualise a full-length segment 3 RNA. Odd numbers show the products of a reaction mix containing reverse transcriptase (RT) while even numbers lack reverse transcriptase. (C) Reverse transcriptase-PCR of the vaccine passaged once at limiting dilution in embryonated hen’s eggs showing full-length segments A1–3. The expected sizes of the PCR fragments derived from the full-length RNAs are: A1 2239 nts, A2 2318 nts, A3 2184 nts, B1 2235 nts, B2 2306 nts, and B3 2235 nts. L, indicates a ladder of markers with the size indicated in nucleotides.