The chikungunya virus (CHIKV) is a positive-sense single-stranded RNA virus of the genus Alphavirus and family Togaviridae. CHIKV has a long history of recurring epidemics (1). CHIKV is primarily transmitted through Aedes mosquitoes, specifically Aedes aegypti and Aedes albopictus, which act as vectors for the virus. Regional epidemics of CHIKV occurred mainly in Africa and Southeast Asia after its identification (2–4). CHIKV infection has an initial stage lasting 2-7 days and is characterized by increased body temperature, vomiting, diarrhea, and joint pain. Post-acute symptoms persist for up to 21 days, and a chronic or arthritic phase may occur in elderly individuals caused by viral encephalitis (1) (2). During the 2005-2007 epidemics in the Indian Ocean islands and India, there were recorded cases of deaths, viral encephalitis, and newborn illnesses connected with CHIKV (3). The length of the CHIKV genome is ~12 kb, including two untranslated regions known as 5’UTR and 3’UTR. CHIKV genome also contains two open reading frames (ORFs) separated by a noncoding junction. The 5’ORF is translated from the genomic RNA (gRNA) that encodes the non-structural polyprotein (P1234) and is later divided into distinct non-structural proteins nsP1 to 4. The 3’ORF translates a positive-sense sub-genomic mRNA (sgRNA) into several structural proteins, including envelope 1 (E1), 6K, envelope 3 (E3), envelope 2 (E2), and capsid (C) (4–7). The rapid spread of CHIKV has increased, and it has become a global pathogen. Being a major public health threat, there is an urgent need for an efficient vaccine. Live-attenuated vaccines are the weaken form or non-virulent form of the virus, reducing its ability to cause illness; similarly, inactivated vaccines render it inactive using chemicals, heat, or UV radiation. These strategies stimulate a robust immune response while ensuring safety and efficacy concerns (8). Subunit vaccines utilize specific virus components, like proteins or peptides, to trigger an immune response, thereby reducing side effects and avoiding total exposure to the virus (9). Vector-based vaccinations use non-pathogenic viruses or bacteria to deliver viral antigens into cells, mimicking a genuine infection to stimulate a potent immune response. Progress in vaccine candidates against chikungunya relies on continuous research and development. Recent advancements have qualified specific candidates for clinical trials, emphasizing the need for diverse immunization strategies (10). The FDA recently authorized Valneva’s single-shot live-attenuated chikungunya vaccine, Ixchiq/VLA1553, on November 9, 2023. This is the only licensed human vaccine against CHIKV; however, several CHIKV candidate vaccines are in clinical trials (11). The lack of long-term monitoring in chikungunya-prone regions such as Africa, Latin America, and Southeast Asia, along with intermittent transmission and unpredictable outbreaks, hinders randomized controlled efficacy studies. This has led authorities to explore alternative data collection methods for developing and authorizing CHIKV vaccines and medical devices (12, 13). The CHIKV E2 surface protein is used in vaccine preparation because the E2 protein is the primary target of neutralizing antibodies, facilitating the virus’s binding to cell membrane receptors and attachment factors. The E2 glycoprotein binds to the Mxra8 membrane receptor, triggering clathrin-mediated endocytosis of CHIKV. Infected individuals develop antibodies that target the E2 protein, which monoclonal antibodies can neutralize. In adult and neonatal mice, anti-E2 antibodies from recovering patients can reduce or eliminate CHIKV infection (4, 5, 7).

Escherichia coli is a versatile bacterium that is a crucial part of human gut microbiota and is being used in recombinant DNA technology for cloning. However, due to its high adaptability, E. coli can cause severe infections. Many strains of E. coli use virulence factors that affect various cellular functions, leading to intestinal and extraintestinal diseases. Despite its potential danger, E. coli remains a valuable laboratory organism (14). E. coli bacteria colonize human infants’ gastrointestinal tracts after birth, coexisting in a symbiotic state. They rarely cause illness unless the immune system weakens or regular gastrointestinal tract barriers are breached. The mucous layer of the mammalian colon is the habitat for commensal E. coli, the predominant facultative anaerobe of the human gut microbiota. The mechanisms behind this symbiotic relationship remain unclear, but a hypothesis suggests it uses gluconate in the colon (15). Some aggressive E. coli strains have acquired characteristics that allow them to adapt to new environments and cause a broader range of diseases. The pathotypes that can infect healthy individuals are limited to the most efficient combinations of virulence factors. These pathotypes can lead to enteric/diarrheal disease, urinary tract infections (UTIs), or sepsis/meningitis. The six distinct families of intestinal pathogens are Enteropathogenic E. coli (EPEC), Enterohaemorrhagic E. coli (EHEC), Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC), and Diffusely Adherent E. coli (DAEC) (16). Urinary tract infections and sepsis are primarily caused by Extraintestinal pathogenic E. coli (ExPEC), a multidrug-resistant strain, leading to treatment failure and hospitalization costs. Adequate immunization could reduce illness and mortality rates, potentially decreasing the bacteria’s prevalence in both healthy and sick populations. Genomic analysis revealed the gene sinH is prevalent in several invasive ExPEC phylogroups (17). ETEC is a genetically diverse pathogen causing millions of symptomatic infections annually, primarily affecting children in low and middle-income countries. Initially discovered 40 years ago, ETEC contributes to the death rate among children under five and causes severe diarrhea. Developing effective vaccinations to prevent ETEC infections remains crucial, but the inherent genetic variability of E. coli poses a significant challenge (18). E. coli strain O157:H7, a zoonotic enteric pathogen, has the potential to induce life-threatening hemolytic uremic syndrome (HUS) because of Shiga toxins (Stx). Additionally, it can lead to severe infections that result in chronic renal failure in children. Hemorrhagic colitis and diarrhea are two commonly recognized symptoms of infection (19). E. coli pathogenic strains cause damage to mucosal surfaces through a sequential pathogenesis process, including colonization, evasion of the host’s immune system, proliferation, and harm. While most E. coli strains remain outside cells, EIEC is a genuine intracellular pathogen capable of invading and replicating within macrophages and epithelial cells. Some strains can enter epithelial cells but do not replicate inside them (14). Developing vaccines for E. coli is complex due to the diverse strains and virulence levels. Researchers aim to create safe and effective vaccines that provide broad protection against various strains by focusing on common targets, shared antigens, and genetic diversity, ensuring a comprehensive strategy to address the diverse pathogenic properties of these strains. This work mainly focuses on preparing an E. coli-based recombinant protein vaccine to protect against viral (CHIKV) and bacterial (E. coli) infection.

The pET28a-E2 plasmid was modified to remove 6-His-Tag by Gibson assembly cloning ( Figure 1A ), and the pET28a-E2 plasmid was transformed into E. coli DH5α bacterial cells. Clones were confirmed by restriction digestion ( Figure 1B ) and DNA sequencing. The pET28a-E2 plasmid was transformed into E. coli Rosetta Bl21 cells and cells were induced by 1mM IPTG for CHIKV E2 protein expression. A western blotting experiment was carried out to confirm CHIKV E2 protein expression in Rosetta Bl21 cells. Western blotting results confirmed CHIKV E2 protein expression with a band of expected molecular weight 47 kDa ( Figure 1C ). Purified CHIKV E2 protein showed protein bands at the same range of 57 kDa, but the control E. coli bacteria did not do so.

CFU method was used to calculate the bacterial number ( Figures 1DI–III ). The E. coli Rosetta Bl21 expressing CHIKV E2 protein bacteria concentration was 1.05×10¹⁰ CFU/ml ( Figure 1DI ), and the E. coli Rosetta Bl21 (control strain) concentration was 2.2×10¹¹ CFU/ml ( Figure 1DII ). Bacterial cells were killed by heating at 70°C for 1 hour. Bacterial cell death was confirmed by platting on an LA plate and incubated for one week at 37°C. No bacterial colonies were observed on an LA plate ( Figure 1DIII ).

The E. coli Rosetta Bl21 expressing CHIKV E2 protein and control E. coli Rosetta Bl21 were used for mouse immunization. For mouse immunization, 1×10⁸ bacterial cells were used for both E. coli Rosetta Bl21 expressing CHIKV E2 protein and E. coli Rosetta Bl21 strains.

The mice were divided into three groups. The first group received E. coli Rosetta Bl21 expressing E2 protein with alum. The second group received E. coli Rosetta Bl21 bacteria (control) with alum. The ratio of bacteria to alum was 1:1. The third group received E. coli Rosetta Bl21 expressing E2 protein without alum. All groups received 1×10⁸ bacteria subcutaneously.

A thirty-day gap was kept between the two doses of immunization. Then, blood was collected by bleeding through the retro-orbital route, and serum was isolated and stored at -20°C for further use. ELISA test was performed to measure the mouse IgG immune response against CHIKV and E. coli. CHIKV was coated in an ELISA plate for determining the mouse IgG immune response against CHIKV.

We observed that E. coli Rosetta Bl21 expressing E2 protein with alum (OD value 0.244 after 2^nd dose) showed a two-fold higher IgG immune response against CHIKV in comparison to E. coli Rosetta Bl21 expressing E2 protein without alum (OD value 0.118 after 2^nd dose) ( Figure 2A ) in male mice. E. coli Rosetta Bl21 expressing E2 protein with alum showed a five-fold higher IgG immune response than control group E. coli with alum (OD value 0.056 after 2^nd dose) in male mice ( Figure 2A ).

Similar results were observed for female BALB/c mice ( Figure 2B ), wherein E. coli Rosetta Bl21 expressing E2 protein with alum (OD value 0.238 after 2^nd dose) showed more than two-fold higher IgG immune response against CHIKV in comparison to E. coli Rosetta Bl21 expressing E2 protein without alum (OD value 0.095 after 2^nd dose), and four-fold higher IgG immune response than control group E. coli (OD value 0.062 after 2^nd dose). The results confirmed that the E2 protein-expressing strain with alum exhibited a significantly higher IgG immune response against CHIKV (p<0.005) than the E2 protein-expressing strain without alum and the control E. coli strain.

E. coli Rosetta Bl21 was coated in an ELISA plate to determine the mouse IgG response against E. coli. Figures 2C, D represent the IgG immune response of different formulations against E. coli. A significantly higher IgG triter was observed for all formulations in both male ( Figure 2C ) and female ( Figure 2D ) immunized mice against E. coli. E2 protein-expressing strain with alum, E2 protein-expressing strain without alum, and the control E. coli strain exhibited OD values of 5.402, 5.517, and 5.5, respectively, after 2^nd dose in male mice ( Figure 2C ). On the other hand, E2 protein-expressing strain with alum, E2 protein-expressing strain without alum, and the control E. coli strain exhibited OD values after 2nd dose of 5.736, 5.37, and 5.538, respectively, after 2nd dose in female mice ( Figure 2D ).

Anti-mouse IgG1 was coated in an ELISA plate to determine the mouse total IgG1 response. Figures 3A, B represent the total IgG1 immune response. A significantly higher IgG1 triter was observed for all formulations in both male ( Figure 3A ) and female ( Figure 3B ) immunized mice. E2 protein-expressing strain with alum, E2 protein-expressing strain without alum, and the control E. coli strain exhibited OD values of 2.85, 3.17, and 2.78, respectively, after 2nd dose in male mice ( Figure 3A ), however the preimmune blood Total exhibited total IgG1 OD values of 0.55, 1.55, and 1.48. Which means that the E2 protein-expressing strain with alum injected mice group showing significant higher IgG1 response then the others groups in male mice. On the other hand, E2 protein-expressing strain with alum, E2 protein-expressing strain without alum, and the control E. coli strain exhibited OD values after 2nd dose of 3.21, 2.5, and 1.86, respectively, in female mice ( Figure 3B ).

We also measured the total IgG2b immune response and observed that E2 protein-expressing strain with alum, E2 protein-expressing strain without alum, and the control E. coli strain exhibited OD values of Exhibited OD value 2.83, 2.4 and 2.55 after 2nd dose ( Figure 3C ) in male mice. On the other hand, E2 protein-expressing strain with alum, E2 protein-expressing strain without alum, and the control E. coli strain exhibited OD values after 2nd dose of 2.78, 2.71, and 2.64, respectively, after 2nd dose in female mice ( Figure 3D ).

Pooled serum samples (male and female mice) from E2 protein-expressing strain with alum and controlled strain with alum, immunized mice serums were used for further analysis. CHIKV in vitro neutralization assay was performed using TCID50 assay. CHIKV viruses were treated with mouse-immunized serum for one hour at 37°C for neutralization. Then, Vero cells were infected by serum-treated CHIKV viruses with inverse dilution from 10^–1 to 10^–10 virus.

At serum dilution 1:100, we observed that CHIKV viruses treated with E2 protein-expressing strain with alum-injected mice serum showed a one-fold decrease in TCID50 value at 12 hours from other groups. Further, the same group showed a two-fold reduction in the TCID50 value ( Figure 4A ) after 120 hours from other groups. When the serum dilution was 1:50, there was a two-fold decrease after 12 hours and a three-fold reduction after 120 hours in TCID50 value compared to both E. coli Rosetta Bl21 with alum (control) and positive CHIKV infection ( Figure 4B ).

During the ongoing COVID-19 pandemic, the whole world faced a crisis. More than 776 million people were infected, and 7.1 million people died of coronavirus infection (https://data.who.int/dashboards/covid19). Twelve COVID-19 vaccines approved by WHO for emergency were used to generate protection among the world population. Out of the twelve vaccines, two were mRNA-based vaccines, four were non-replicating viral vector-based vaccines, three were inactivated virus-based vaccines, and three were recombinant vaccines (https://covid19.trackvaccines.org/agency/who/). During the pandemic, nucleoside-modified (mRNA and non-replicating viral vector-based vaccines) vaccines came first because of the less time needed to synthesize the vaccine. Synthetic mRNA, a therapeutic component of vaccines, has been used for over 30 years. However, limited research exists on its distribution in the body, cell absorption, translation, and functional lifespan. A recent study found that mRNA can reverse the transcription of vaccine sequences in transfected cells, causing potential safety issues (24).

The continuous presence of NMS-mRNA in the cytoplasm disrupts the removal process, activating natural transposable elements (TEs) and triggering a coordinated innate immune response against foreign genetic material. This can lead to auto-inflammatory and autoimmune disorders and harmful mutations in reverse-transcribed molecules, potentially causing DNA damage. Understanding the intracellular processes triggered by mRNA absorption is crucial for effective vaccine development (24). mRNA vaccines are vital in vaccination campaigns, but they can cause adverse effects due to the pro-inflammatory properties of lipid nanoparticles or mRNA. Further investigation into these mechanisms is needed to ensure safety, build trust, and inform health regulations, despite existing understanding primarily from cell experiments (25). Therefore, in the COVID-19 period, the vaccine was the most critical priority. However, protein-based vaccines are safer to use as vaccines.

Nevertheless, the problem associated with protein-based vaccines is the purification of protein through HPLC, size-based chromatography, and ion-exchanged chromatography. Further, it takes time to optimize protein purification and confirmation. In this study, our findings suggest a faster, safer, and cost-effective recombinant protein-based vaccine preparation technique that can protect against both viral and bacterial infection. The results show that our advanced technique generates an IgG immune response against CHIKV and a robust IgG immune response against E. coli. The IgG1 is the primary IgG immunoglobulin in mice. IgG1 is primarily associated with a Th2 based immune response and activate CD8⁺ T-cells. The higher IgG1 immune response shown on E2 protein-expressing strain with alum mice groups. IgG1 can interact with FcγRs on dendritic cells (DCs), leading to T cell activation and cytokine synthesis. IgG1 can bind to FcγRs on dendritic cells, specifically FcγRI and FcγRII. This binding results in the internalization of antigen-IgG complexes, known as immune complexes (ICs). The internalization of immunological complexes by dendritic cells stimulates Fc gamma receptors, thereby augmenting endosomal maturation and lysosomal fusion. This improves antigen processing. DCs process antigens for presentation on major histocompatibility complex (MHC) class II molecules. This presentation activates antigen-specific T cells (26–29). In mice, IgG2b antibodies are mostly associated with a Th1-type immune response that activates CD4⁺ T cells. IgG2b engages with activating Fc receptors on immune cells, particularly FcγRIV, which is essential for triggering a Th1 response (30, 31). The in vitro neutralization assay against CHIKV showed three-fold and two-fold viral neutralization against control in 1:50 and 1:100 serum dilution, respectively. Our vaccine preparation method/technique is easy to perform, and there is a minimum chance of post-purification protein miss-folding because recombinant protein is present within the cells.

Another problem with vaccine development against infectious agents like viruses and bacteria is the working person, laboratory leakage, and safety concerns. A biosafety laboratory is mandatory to work against infectious agents, and the cost of preparing and maintaining this laboratory is very high. During the COVID-19 pandemic, the world faced testing of samples and research facilities because of insufficient biosafety laboratory systems. Also, expertise, large-scale production, deployment, and storage were significant challenges (32). The cost associated with biosafety facilities also affects vaccination programs directly. Therefore, our approach of recombinant protein-expressing bacteria as a candidate vaccine can quickly solve this problem. This can be due to fewer biosafety concerns, low-cost production, easy handling, and rapid vaccine production.

Furthermore, this bacteria-expressing viral protein vaccine can be stored at −20°C. Thus, the storage cost can be significantly reduced. This approach can also be used to immunize humans against pathogenic bacteria using non-pathogenic animal bacteria. For example, Salmonella sp., which causes fowl typhoid and is non-pathogenic to humans, can be used for immunization against typhoid and other infectious diseases. Further research in E. coli-based recombinant protein vaccines could help prepare vaccines that protect from multiple infections.