Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. By two years of age, more than 90% of children have been infected at least once with this virus. RSV infections typically cause mild illness; however, severe respiratory disease can occur and is associated with symptoms such as bronchiolitis and wheezing. RSV infection is also common in the elderly, in whom it can be deadly. Despite the enormous impact of RSV on human health, the design of a safe and successful vaccine has eluded researchers.

During the 1960s, researchers thought they had a vaccine. But they were wrong—the experimental, formalin-inactivated RSV vaccine administered to children not only failed protect them but also exacerbated their illness in response to natural RSV exposure. Many of the children developed bronchiolitis and pneumonia so severe that it required hospitalization, and, unfortunately, two of the vaccinated children died1.

The formalin-inactivated RSV vaccine did elicit antibodies—but these were not neutralizing, meaning that they did not inactivate the virus. High titers of RSV were recovered from the lungs of the two children that died, demonstrating the ineffectiveness of the vaccine in eliciting a protective immune response1.

In this issue of Nature Medicine, Delgado et al.2 provide insight into the failure of the formalin-inactivated RSV vaccine. They report that inactivated forms of RSV do not sufficiently activate various pattern recognition receptors, molecules important for the recognition of pathogens by immune cells and expressed by several cell types including B cells, the cells that produce antibodies. As a result of this poor activation, B cells produce only low-avidity antibodies that poorly recognize the virus. Because the vaccine elicited such poor antibodies, it failed to inhibit virus replication and resulted in enhanced disease, probably caused by the host immune response.

The vaccine-enhanced disease exhibited by the children during the 1960s vaccine trial can be recapitulated in mice. Early studies in the mouse model clearly established that the host immune response contributes to the development of RSV vaccine–enhanced disease. As in people, immunization with formalin-inactivated RSV results in the production of non-neutralizing RSV-specific antibodies3. After RSV challenge, these non-neutralizing antibodies cause the formation of immune complexes consisting of antibodies bound to the surface of the RSV virion4.

Previous work from this group showed that complement components activated by these immune complexes were necessary for bronchoconstriction during RSV infection of mice previously immunized with the vaccine5. What has been unclear is why immunization results in the production of these nonprotective antibodies, impeding the development of new RSV vaccines for decades.

One hypothesis is that formalin treatment disrupts the folding of proteins on the surface of the RSV virion that harbors protective antibody epitopes—accounting for the inability to induce a neutralizing antibody response3,6,7. The results obtained by Delgado et al.2 suggest that the situation is a bit more complicated.

Delgado et al.2 compared the antibody response after immunization with either live RSV or inactivated forms of the virus. As expected, inactivated forms of the virus elicited only low-avidity antibodies, whereas live RSV induced neutralizing antibodies of high avidity. Immunization with the inactivated forms of RSV resulted in increased airway hyperresponsiveness and increased virus titers in the lung after RSV challenge as compared to mice previously vaccinated with live virus.

A number of Toll-like receptors (TLRs) and other pattern recognition receptors are activated by RSV infection. The fusion protein of RSV is recognized by TLR4 on the cell surface8, the RSV single-stranded RNA genome is recognized by TLR7 (ref. 9) and RSV double-stranded RNA is recognized by the helicases retinoic acid–inducible gene-1 (RIG-I) and melanoma differentiation–associated protein-5 (MDA-5)10 expressed inside of host cells (Fig. 1). Delgado et al.2 hypothesized that the inability of inactivated forms of RSV to replicate compromised their ability to activate pattern recognition receptors, resulting in less efficient B cell stimulation.

Figure 1: Differential activation of pattern recognition receptors by live RSV versus formalin-inactivated RSV.
figure 1

Katie Ris-Vicari

The F protein of RSV is capable of being recognized by TLR4 expressed on the cell surface of host cells. RSV typically enters the host cell through fusion at the plasma membrane. In some cases, virus entry can occur via receptor-mediated endocytosis of RSV virions coated with antibodies. Once inside the cell, a number of host cell pattern recognition receptors are capable of recognizing virus-derived nucleic acids. RIG-I and MDA-5 can recognize double-stranded RNA (dsRNA) intermediates, whereas TLR7 is activated by virus-derived single-stranded RNA (ssRNA). The combined stimulation of TLR4, TLR7, RIG-I and/or MDA-5 by live RSV infection leads to the activation of multiple downstream signaling pathways that ultimately results in full activation of the B cell and is necessary to achieve antibody affinity maturation. Delgado et al.2 demonstrate that formalin-inactivated RSV fails to engage and activate pattern recognition receptors found inside the host cell, such as TLR7, RIG-I and MDA-5, because of the failure of the virus to replicate. Therefore, only TLR4 is sufficiently stimulated after formalin-inactivated RSV vaccination. The B cells fail to produce high-avidity antibodies in the absence of sufficient TLR stimulation.

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They initially tested this by examining mice deficient in myeloid differentiation factor-88, an adaptor molecule used by several pattern recognition receptors to mediate signals2. They showed that RSV infection of myeloid differentiation factor-88–deficient mice resulted in the production of lower avidity antibodies specific to RSV2. Finally, they found that stimulation of TLRs increased the avidity of the antibodies elicited after immunization with inactivated forms of RSV2. Mice treated this way had lower airway hyperresponsiveness and lower virus titers in the lung after RSV challenge infection.

These findings implicate low-avidity antibodies in the development of RSV vaccine–enhanced disease. But there are probably other events that also contribute to disease. Previous work has shown that CD4+ T cells are required to induce pneumonia after RSV challenge of mice previously immunized with formalin-inactivated RSV11. Moreover, recent research has indicated that formalin-inactivated RSV fails to elicit a RSV-specific CD8+ T cell response12.

Taken together, these studies indicate that immunization with formalin-inactivated RSV elicits a low-avidity, RSV-specific antibody response, a robust RSV-specific CD4+ T cell response and no cytotoxic CD8+ T cell response. The exacerbated disease in the children was probably due to these unique circumstances that together created the perfect storm. A strong, RSV-specific CD4+ T cell response would have been maintained for a longer time in the face of increased virus titers, owing to the absence of an effective cytotoxic CD8+ T cell response and an ineffective antibody response.

The findings could have broader implications for vaccine development, as inappropriate immune responses elicited by vaccination are not limited to RSV. For instance, immunization with an experimental formalin-inactivated measles virus vaccine resulted in an increased incidence of atypical measles infections among the vaccinees13. A number of effective formalin-inactivated virus vaccines are currently in use, suggesting that the relative balance between a protective versus pathogenic immune response elicited by vaccination with a formalin-inactivated vaccine is dependent on the nature of the protective immunity required for a particular pathogen.

Future studies aimed at dissecting out the distinct roles of antibodies and T cells in mediating RSV vaccine–enhanced disease are essential for gaining a further understanding of the failure of the formalin-inactivated RSV vaccine. The intriguing results obtained by Delgado et al.2 suggest that the addition of TLR-stimulating adjuvants may conceivably be enough to make a safe and effective vaccine, but we need to know more before such a treatment can become a reality.