High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector

Area of Science:

• Vaccinology
• Molecular Biology
• Immunology

Background:

• Replication-incompetent adenovirus type 35 (rAd35) is a promising vaccine carrier due to its ability to induce strong T- and B-cell responses.
• Unlike adenovirus type 5 (Ad5), Ad35 is rare in human populations, minimizing pre-existing neutralizing antibodies.
• rAd35 is being explored for vaccines against malaria, AIDS, and tuberculosis.

Purpose of the Study:

• To engineer rAd35 vectors capable of carrying multiple vaccine antigens within a single vector.
• To overcome limitations of single-antigen delivery systems for complex diseases.
• To expand the utility of rAd35 as a versatile vaccine platform.

Main Methods:

• Construction of rAd35 vectors with two expression cassettes driven by cytomegalovirus (CMV) promoters.
• Ensuring cassette functionality by utilizing non-identical polyadenylation signals.
• Evaluation of vector stability and transgene expression.
• Assessment of immune responses to model antigens (HIV Env-gp120 and Plasmodium falciparum CSP) expressed from the dual-cassette vector.

Main Results:

• Successfully generated stable rAd35 vectors containing two expression cassettes.
• Demonstrated that transgene expression is unaffected by the presence of a second cassette.
• Confirmed that non-identical polyadenylation signals are crucial for dual-cassette functionality.
• Showed that potent T- and B-cell responses are induced against both model antigens from a single rAd35 vector.

Conclusions:

• Engineered dual-cassette rAd35 vectors are stable and functional.
• These vectors efficiently express multiple antigens and elicit robust, antigen-specific immune responses.
• The developed rAd35 platform significantly enhances vaccine development potential for complex diseases like malaria, AIDS, and tuberculosis.