In vitro studies show peroxide-inactivated candidates stimulate robust CD8+ T-cell and antibody responses.

Scientists at Oregon Health & Science University working with colleagues at the spin-out firm Najit Technologies published in vivo data demonstrating the utility of a hydrogen peroxide (H2O2)-based approach to generating inactivated viral vaccines. Studies in mice demonstrated that H2O2-inactivated viral vaccines against lymphocytic choriomeningitis virus (LCMV), smallpox, and even West Nile virus triggered high titres of virus-specific CD8+ T cells or neutralizing antibodies and provided long-term protection against subsequent challenge with lethal doses of the respective pathogens.

Mark K. Slifka, Ph.D., Ian J Amanna, Ph.D., Hans-Peter Raué, Ph.D., and colleagues report their studies in Nature Medicine in a paper titled “Development of a new hydrogen peroxide–based vaccine platform.”

The standard approach to preparing inactivated viral vaccines is based on the use of formaldehyde or β-propiolactone (BPL). However, formaldehyde can damage viral epitopes leading to reduced immunogenicity. In some cases this can even result in serious exacerbation of disease following infection. BPL inactivation, meanwhile, can trigger unwanted immune responses including allergic reactions through chemical modifications of vaccine components.

The researchers first confirmed that the oxidizing agent H2O2 effectively inactivates both DNA and RNA viruses. They then demonstrated that the peroxide inactivation didn’t impact greatly on immunogenicity. When yellow fever virus (YFV) inactivated using either formaldehyde, BPL, or H2O2 was probed with immune serum from infected mice, the H2O2-inactivated virus retained 87–98% of the maximum antibody binding response observed with live virus. In contrast, YFV inactivated using formaldehyde or BPL demonstrated markedly reduced immunogenicity.

The researchers then moved on to test H2O2-inactivated viral vaccines in vivo. They used the oxidizing agent to inactivate lymphocytic choriomeningitis virus (LCMV) and immunized experimental mice with the resulting purified vaccine candidate. Protective immunity to LCMV is mediated in vivo by CD8+ T cells, and the peroxide vaccine-treated animals were found to generate levels of LCMV-specific CD8+ T-cell responses equivalent to those produced by mice given a live recombinant vaccinia virus (VV) vaccine expressing LCMV nucleoprotein or glycoprotein.

Importantly, while CD8+ T cells from both cohorts of vaccinated mice also expressed interferon-γ after subsequent stimulation with LCMV peptide ex vivo, CD8+ T cells taken from the H2O2 LCMV vaccine-immunized mice more efficiently co-expressed interferon-α (IFN-α) and were also 10 times better at producing interleukin-2 after peptide stimulation. “51 ± 5% of IFN-γ+ CD8+ T cells from H2O2-LCMV–vaccinated mice co-expressed IL-2, compared with just 3 ± 1% of virus-specific T cells from LCMV-Armstrong-infected mice,” the authors write.

To see how well the H2O2-LCMV vaccine-induced CD8+ T cells proliferated in vivo, vaccinated animals were challenged with live LCMV-Armstrong 28 days after vaccination. Virus-specific CD8+ T-cell counts were measured just before viral challenge and four days after challenge. When compared with live virus-vaccinated animals, mice given the H2O2-LCMV vaccine generated double the frequency of NP118 protein-specific CD8+ T cells (equivalent to 22 ± 6% of the total CD8+ T-cell compartment) than live mice-vaccinated animals (10 ± 2%).

The H2O2-LCMV vaccine was similarly able to protect mice infected with an LCMV clone (clone 13) known to cause chronic infection in naive mice. The immunized mice exhibited either complete or more than 99% clearance of the virus within seven days. “Together, these results demonstrate that multifunctional CD8+ T cells can be elicited with a H2O2-inactivated vaccine and provide protective immunity against chronic viral infection,” the authors state.

To see whether H2O2 inactivation could generate a vaccine that elicits neutralizing antibody responses the researchers turned to smallpox vaccination. They gave mice subcutaneous injections of either a live attenuated smallpox vaccine (consisting of VV), a live modified vaccinia Ankara vaccine (which is replication deficient in humans and mice), or purified VV inactivated using either H2O2, formaldehyde, or ultraviolet (UV) irradiation. Antibody responses were measured 28 days after booster vaccination.

Again, not only did the H2O2-VV vaccination elicit much greater virus-specific neutralizing antibody responses than the vaccines generated using other inactivation approaches, but levels of antibody response induced by the H2O2-VV vaccine also more closely mirrored the immunity induced by live viral infection. And when immunized animals were subsequently challenged with a lethal dose of VV, 100% of the H2O2-VV-vaccinated animals survived.

Similarly, mice vaccinated with an H2O2-inactivated West Nile virus (WNV) vaccine generated 10-fold higher virus-specific antibody titers after booster vaccination than mice given Ft. Dodge Innovator, a formaldehyde-inactivated WNV vaccine used in horses. Encouragingly, in the experimental mice, antiviral antibody titers peaked at about 28 days after booster vaccination with the H2O2-WNV vaccine and showed no evidence of declining over the next six months of observation.

In fact, H2O2-WNV provided experimental animals with full protective immunity to a lethal dose of WNV administered more than 280 days after they were immunized. Of particular note was the finding that immune serum from H2O2-WNV-vaccinated animals was sufficient to completely protect naive mice against challenge with WNV, indicating that protective immunity to the virus can be mediated by neutralizing antibodies.

The researchers admit that extrapolating these findings to humans isn’t easy and it isn’t known what level of immunity is needed to protect humans against WNV. However, they point out, “those who survive WNV infection are believed to be immune for life, and H2O2-WNV vaccination of mice elicits antibody responses that seem to meet or exceed the levels of immunity that develop after natural infection in humans.

“We have found that H2O2 represents a new and versatile platform for the development of inactivated vaccines,” the investigators conclude. “Exposure to H2O2 resulted in minimal damage to viral epitopes and provided improved antigenicity and immunogenicity when compared to other standard approaches used for virus inactivation. The ability of H2O2 to inactivate a wide spectrum of pathogens including viruses, bacteria, parasites, and potentially even bacterial spores may allow expansion into new areas of vaccine research, fulfilling previously unmet needs for combating a number of human pathogens.”

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