DNA Cocktail Vaccines

• Chimeric DNA Vaccines refer to the antigenic form of expressed proteins
• The module covers Xenogeneic DNA vaccines, DNA vaccines with genetic adjuvants, DNA cocktail vaccines, multigene vaccines, and DNA vaccines with immune stimulatory sequences and costimulatory molecules
• The module runs for 6 hours

DNA Cocktail Vaccines

• These vaccines are a combination of multiple DNA vaccines
• These contain different antigens of interest
• They induce an enhanced immune response in the host
• DNA vaccines are delivered into the body, expressing genes of interest
• The expressed genes prompt the body to produce antigens
• The body's immune system creates a humoral and cellular immune response

Introduction to TB vaccines

• The global TB epidemic persists due to the lack of a highly effective vaccine
• The current TB vaccine, Mycobacterium bovis BCG, has variable efficacy in controlled clinical trials
• There are doubts about it's value in protecting against adult pulmonary TB
• Its effectiveness in preventing TB in HIV-infected individuals is uncertain
• It's a live vaccine, and cases have been reported of it re-activating in HIV infected individuals
• BCG vaccination is not indicated for immunocompromised individuals

DNA vaccine cocktail for TB

• Dr.Morris laboratory generated a DNA cocktail expressing mycobacterial proteins fused at the N terminus to Ubiquitin (UB)
• Ubiquitin serves as a eukaryotic intracellular targeting sequence
• These Ubiquitin-conjugated TB proteins enhance major histocompatibility complex class I presentation

Ubiquitin

• It is a 76-amino-acid peptide that controls normal protein intracellular turnover in eukaryotic cell cytoplasm
• Proteins to be degraded are tagged with ubiquitin molecules and targeted to the proteasome system
• Ubiquitin conjugation enhances proteasome-dependent degradation of synthesized antigens
• It increases the cell-mediated response induced in vivo against the conjugated antigen

Ubiquitin Signaling in Immune Responses

• Ubiquitination posttranslational mechanism regulates signal transduction in diverse biological processes
• It regulates pattern-recognition receptor signaling
• This mediates both innate immune responses and dendritic cell maturation for adaptive immune responses
• Ubiquitination regulates T cell development, activation, and differentiation
• This helps maintain efficient adaptive immune responses to pathogens and immunological tolerance to self-tissues
• Ubiquitination is a reversible dynamic reaction controlled by ubiquitin ligases (E1, E2 & E3) and deubiquitinases (DUB)
• Deregulated ubiquitination events are associated with immunological disorders

Protein degradation

• The best-known function of ubiquitination is targeting substrate proteins for degradation in the 26S proteasome
• Some ubiquitin chains signal transduction via nondegradative mechanisms
• Such ubiquitin chains facilitate protein-protein interactions in signal transduction
• The 26S proteasome is a large, multi-catalytic ATP-dependent protease complex
• It degrades the arm of the ubiquitin system
• It's the major pathway for regulated degradation of cytosolic, nuclear, and membrane proteins in all eukaryotic organisms

Life cycle of the proteasome

• The "birth" of the proteasome is controlled by transcriptional regulation of its subunits
• The biogenesis is organized by transcription factors
• These include Nrf1 and Rpn4, which are sensitive to changing physiological conditions
• The subunits assemble in a coordinated manner to form the mature proteasome
• The 26S proteasome recognizes ubiquitin conjugated substrates using intrinsic and extrinsic ubiquitin receptors.
• Recognition is regulated by post-translational modifications, disassembly, conformational changes, and cellular localization
• The "death" of the proteasome is mediated by the lysosome/vacuole/autophagy and caspase(s)
• Energy-dependent ubiquitin-substrate conjugate formation is catalyzed by E1, E2, and E3

DNA Vaccine Cocktail Study

• A study showed Immunization with a DNA Vaccine Cocktail Protects Mice Lacking CD4 Cells against an Aerogenic Infection with Mycobacterium tuberculosis
• Tuberculosis (TB) is the most common opportunistic disease and a potentially fatal complication among immunocompromised individuals infected with human immunodeficiency virus (HIV)
• They showed that vaccination of CD8–/– mice with a TB DNA vaccine cocktail didn't enhance protective responses to a Mycobacterium tuberculosis infection
• Immunization with a DNA vaccine cocktail, or BCG, induces antituberculosis protective immunity regardless of CD4 cells
• In vaccinated CD4-/- animals, bacterial burdens in organs were reduced and the lung pathology has improved

Ubiquitin fusion protein constructs

• Protease complex (PC) should cleave the UbG76 chimeric protein and release the mature antigen.
• UbA construct exploits the reduced ability of the protease complex to cleave at position A76, leaving the intact UbA molecule a target for rapid polyubiquitination.
• Ub conjugation for the UbGR construct adds an arginine to the N terminus of the mycobacterial antigen, destabilizing the protein and enhancing the degradation rate

Results on specific protein constructs

• The constructs expressing relatively stable antigens (tPA64 and UbG64) induced substantial humoral responses and only moderate levels of IFN-γ
• Expression of the less-stable UbA64 fusion protein after DNA vaccination yielded a weaker, but Th1-polarized, humoral response and substantial cytokine production
• Immunization with the plasmid encoding the UbGR64 chimera generated a robust IFN- y response
• There were no anti-MPT64 antibodies with such immunization
• If the a R64 protein is rapidly and completely degraded intracellularly, there is insufficient intact protein to interact with B cells
• If targeting the endogenously synthesized Ub fusion protein to the proteasome for cleavage results in effective antigen presentation it can induce a highly polarized Th1 type immune response
• Expression of other TB antigens as UbA or UbGR fusion proteins may yield protective immunity against M. tuberculosis challenge
• The post-challenge mean times to death of vaccinated CD4-/- mice was extended when using a DNA cocktail or BCG
• The substantial CD8-mediated protective immunity in the absence of CD4 cells suggests it may be possible to develop effective TB vaccines for use in HIV-infected populations

Components of the TB DNA cocktail:

• The TB DNA cocktail consists of eight individual vaccines expressing TB antigens
• The TB antigens are:
  • antigen 85B
  • ESAT-6
  • KatG
  • MPT8.4
  • MPT12
  • MPT63
  • MPT64
  • MPT83
• The components were prepared by combining equal amounts of the plasmids to a final concentration of 1 mg of DNA/ml in phosphate-buffered saline (PBS)
• Mice were injected intramuscularly in all four limbs on days 1, 21, and 42 with 200 µg of the cocktail or a single plasmid vaccine
• Five weeks after the final DNA vaccination, the mice were challenged aerogenically with 200 CFU of M. tuberculosis

Results in vaccinated mice

• Immunization with a TB DNA cocktail stimulates CD4+ and CD8+ T cells in wildtype mice
• WT CFU values were typical for this aerogenic infection model
• About 90% reduction (1 log10) in the bacterial burden in the lungs and spleens was seen for WT mice vaccinated
• Relative bacterial growth was reduced at 1 month postchallenge in CD4-/- mice vaccinated
• There were decreases in virulent organisms in the lungs for the DNA cocktail and BCG vaccine groups
• Reductions in splenic CFU for both groups has been detected for both groups of vaccinated CD4-/- mice

Lung features in immunized and non-immunized mice

• When lung tissues from naive CD4-/- mice were stained with the Ziehl-Neelsen stain, acid-fast bacilli were seen
• Infections were subacute and less severe in the CD4-/- mice had been vaccinated
• Lungs had much less inflammation and contained predominantly lymphocytes, with fewer numbers of macrophages
• Ziehl-Neelsen stains of lung tissue from immunized CD4-/- mice revealed substantially smaller numbers of acid-fast bacilli nonvaccinated controls (Fig. 3e and f)
• Lung pathology data at 1 month postchallenge paralleled the bacterial growth results.
• Differences in are seen in lung tissue from immunized with the plasmid cocktail or BCG compared to nonimmunized CD4-/- mice
• Aerogenic tuberculous infection caused severe acute multifocal suppurative pneumonia with extensive necrosis in the nonvaccinated CD4-/-
• Inflammatory cells in these lung sections were predominantly neutrophils, few macrophages and lymphocytes were detected

Immunological Considerations

• Stimulation of APCs from vaccinated animals in the absence of CD4 cells involve TLRs
• Binding of pattern recognition receptors by bacterial products or vaccine components can upregulate the immunostimulatory molecules of APCs
• In this study the direct stimulation of APCs by vaccine components via interactions with TLRs alleviates the necessity for CD4 help
• Recognition of the CpG motifs of DNA vaccines by TLR-9 and the binding of BCG-derived molecules by TLR-2 or TLR-4 elevates the costimulatory signals of APCs
• This is needed to effectively prime naive CD8 T cells

Toxoplasma gondii Study

• Vaccination with a DNA vaccine cocktail encoding Toxoplasma gondii limited protection in mice