Inactivation of multidrug-resistant bacteria and bacterial spores and generation of high-potency bacterial vaccines using ultrashort pulsed lasers.

Multidrug-resistant organisms (MDROs) represent a continuing healthcare crisis with no definitive solution to date. An alternative to antibiotics is the development of therapies and vaccines using biocompatible physical methods such as ultrashort pulsed (USP) lasers, which have previously been shown to inactivate pathogens while minimizing collateral damage to human cells, blood proteins, and vaccine antigens. Here we demonstrate that visible USP laser treatment results in bactericidal effect (≥3-log load reduction) against clinically significant MDROs, including methicillin-resistant Staphylococcus aureus and extended spectrum beta-lactamase-producing Escherichia coli. Bacillus cereus endospores, which are highly resistant to conventional chemical and physical treatments, were also shown to be effectively inactivated by USP laser treatment, resulting in sporicidal (≥3-log load reduction) activity. Furthermore, we demonstrate that administration of USP laser-inactivated E. coli whole-cell vaccines at dosages as low as 105 cfu equivalents without adjuvant was able to protect 100% of mice against subsequent lethal challenge. Our findings open the possibility for application of USP lasers in disinfection of hospital environments, therapy of drug-resistant bacterial infections in skin or bloodstream via pheresis modalities, and in the production of potent bacterial vaccines.

Chemical-free inactivated whole influenza virus vaccine prepared by ultrashort pulsed laser treatment.

There is an urgent need for rapid methods to develop vaccines in response to emerging viral pathogens. Whole inactivated virus (WIV) vaccines represent an ideal strategy for this purpose; however, a universal method for producing safe and immunogenic inactivated vaccines is lacking. Conventional pathogen inactivation methods such as formalin, heat, ultraviolet light, and gamma rays cause structural alterations in vaccines that lead to reduced neutralizing antibody specificity, and in some cases, disastrous T helper type 2-mediated immune pathology. We have evaluated the potential of a visible ultrashort pulsed (USP) laser method to generate safe and immunogenic WIV vaccines without adjuvants. Specifically, we demonstrate that vaccination of mice with laser-inactivated H1N1 influenza virus at about a 10-fold lower dose than that required using conventional formalin-inactivated influenza vaccines results in protection against lethal H1N1 challenge in mice. The virus, inactivated by the USP laser irradiation, has been shown to retain its surface protein structure through hemagglutination assay. Unlike conventional inactivation methods, laser treatment did not generate carbonyl groups in protein, thereby reducing the risk of adverse vaccine-elicited T helper type 2 responses. Therefore, USP laser treatment is an attractive potential strategy to generate WIV vaccines with greater potency and safety than vaccines produced by current inactivation techniques.