The mechanism responsible for lack of protection in the antibody response elicited by FIRSV against RSV remained unclear for decades, hampering development of new vaccines against the virus. This study demonstrates that non-protective antibody elicited by inactivated RSV immunogens, and ultimately associated with the development of ERD(4
), results from lack of affinity maturation due to deficient TLR activation in B cells. These findings modify the previous paradigm, ascribing poor antibody function to formalin disruption of protective epitopes(7
). Also, they open the possibility that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR-agonists in their formulation.
Several observations support the importance of antibody avidity for protection against respiratory viruses, including measles virus(MV) with its risk for atypical disease(21
). A formalin-inactivated vaccine against MV elicited low avidity, non-protective antibody associated with serious clinical manifestations in individuals exposed to wtMV(21
). In this case, low avidity antibody neutralized viral infection through the CD46 low affinity MV receptor, but did not through the CD150 high affinity wtMV receptor and promoted immune complex-mediated illness(21
). However, low avidity polyclonal responses against other viruses may suffice to confer protection. For example, disease enhancement was never described for influenza virus despite wide use of protective non-replicating vaccines that do not promote affinity maturation(39
). The low affinity interaction between the binding site for the receptor in the influenza hemagglutinin and the cellular receptor for the virus (KD=2×10−3
;40) may facilitate neutralization by antibodies of relatively low avidity. In the case of RSV, differences in affinity affect the neutralizing capacity of anti-RSV monoclonal antibodies(15
) and our study provides evidence for their critical role in natural protection against wt infection. The wt receptor for RSV has not been identified. Perhaps, differences in affinity of the interaction between viral attachment proteins and their receptors or in the mechanisms of neutralization among viruses may explain the discrepancies in the efficacy of inactivated vaccines against RSV and influenza(23
This report highlights the importance of TLR activation in B cells for protection against RSV and prevention of ERD. RSV F interacts with TLR4 initiating membrane fusion and MyD88-dependent and independent pathways leading to activation of several transcription factors, notably NF-κB. Once RSV begins transcribing and replicating its genome, TLR-mediated detection appears to depend upon autophagy-associated mechanisms that engage TLR7 and TLR3 in endosomic compartments(cytosolic viral dsRNA also activates CARD-helicases and PKR)(33
). Our data suggest that efficient detection of RSV by TLR, which converge on a redundant set of transcription factors, is necessary for antibody production and affinity maturation. Non-replicating RSV vaccines(e.g.:UVRSV) stimulate only a subset of these receptors in a weak fashion(like TLR4; ref.31
), becoming protective only after incorporation of exogenous TLR ligands. Since most TLR share the same downstream effectors, different agonists may achieve similar effects provided that the intensity of the signal is appropriate. This conclusion is supported by the synergistic effect of adjuvants containing mixed-TLR ligands. Remediation of UVRSV with TLR agonists leads to dendritic cell maturation, T helper activation and B cell affinity maturation. Specifically, our B cell transfer experiments and those using Myd88+/−
mice suggest the existence of a threshold of activation below which affinity maturation does not occur and protective antibody is not elicited. Moreover, for wtRSV the threshold can only be surpassed if MyD88-dependent pathways in B cells are engaged. This does not imply that MyD88-independent pathways do not play a role, but rather that their activation alone is insufficient.
Perhaps a consequence of choosing formalin as the chemical for vaccine inactivation in 1966 was to bias the T cell response in ERD towards Th2(7
). Partial alteration of RSV epitopes during formalin inactivation likely contributed to the generation of non-protective antibody. But epitope disruption played a secondary role in disease priming, as the low avidity antibodies elicited by FIRSV recognized protective epitopes, and other non-replicating RSV vaccines not treated with formalin in this and other publications showed evidence of priming for aberrant immune manifestations(6
). Other studies associated the Th2 bias in ERD to the absence of Th-modulatory CTL in FIRSV recipients(8
) or postulated a role for alum in T cell polarization(47
). However, ERD phenotypes did not differ with or without alum (5
). Whether deficient TLR activation contributed to the Th2 bias in ERD remains to be determined.
Interestingly, our findings may also contribute to clarify why none of the children who were seropositive for RSV before immunization with FIRSV developed ERD(4
), even though wtRSV infections confer only partial protection against subsequent exposures(1
). The pre-existing high avidity antibody elicited by wtRSV before immunization, likely “outcompeted” the low avidity clones elicited by FIRSV and prevented non-protective, pathogenic B cell priming against the virus. Our study also may explain why no child ever developed ERD twice, as the B cells elicited by RSV infection during ERD also eventually “outcompeted” pathogenic B cells primed by FIRSV and reestablished a normal response against future re-infections.
In summary, we demonstrate a critical role for antibody avidity in protective responses against RSV and in the pathogenesis of ERD. Poor TLR stimulation by inactivated RSV vaccines was associated with lack of maturation and led to production of non-protective antibodies. These antibodies were critical for ERD pathogenesis, as they failed to neutralize RSV allowing unrestricted replication and secondary stimulation of FIRSV-primed Th2 cells. Further, low avidity antibody contributed to disease severity through immune complex formation and deposition in affected tissue(5
). Our findings indicate that safe and effective RSV vaccines for infants require neutralizing antibody with similar avidity for protective antigens to that elicited by live virus inoculation. (3000 words)