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A Review of Gene Knockout Strategies for Microbial Cells

Tang PW, Chua PS, Chong SK, Mohamad MS, Choon YW, Deris S, et al.

Recent Pat Biotechnol, 2015;9(3):176-97.
PMID: 27185502

BACKGROUND: Predicting the effects of genetic modification is difficult due to the complexity of metabolic net- works. Various gene knockout strategies have been utilised to deactivate specific genes in order to determine the effects of these genes on the function of microbes. Deactivation of genes can lead to deletion of certain proteins and functions. Through these strategies, the associated function of a deleted gene can be identified from the metabolic networks.
METHODS: The main aim of this paper is to review the available techniques in gene knockout strategies for microbial cells. The review is done in terms of their methodology, recent applications in microbial cells. In addition, the advantages and disadvantages of the techniques are compared and discuss and the related patents are also listed as well.
RESULTS: Traditionally, gene knockout is done through wet lab (in vivo) techniques, which were conducted through laboratory experiments. However, these techniques are costly and time consuming. Hence, various dry lab (in silico) techniques, where are conducted using computational approaches, have been developed to surmount these problem.
CONCLUSION: The development of numerous techniques for gene knockout in microbial cells has brought many advancements in the study of gene functions. Based on the literatures, we found that the gene knockout strategies currently used are sensibly implemented with regard to their benefits.
Fulltext A refined medium to enhance the antimicrobial activity of postbiotic produced by Lactiplantibacillus plantarum RS5

Ooi MF, Foo HL, Loh TC, Mohamad R, Rahim RA, Ariff A

Sci Rep, 2021 Apr 07;11(1):7617.
PMID: 33828119 DOI: 10.1038/s41598-021-87081-6

Postbiotic RS5, produced by Lactiplantibacillus plantarum RS5, has been identified as a promising alternative feed supplement for various livestock. This study aimed to lower the production cost by enhancing the antimicrobial activity of the postbiotic RS5 by improving the culture density of L. plantarum RS5 and reducing the cost of growth medium. A combination of conventional and statistical-based approaches (Fractional Factorial Design and Central Composite Design of Response Surface Methodology) was employed to develop a refined medium for the enhancement of the antimicrobial activity of postbiotic RS5. A refined medium containing 20 g/L of glucose, 27.84 g/L of yeast extract, 5.75 g/L of sodium acetate, 1.12 g/L of Tween 80 and 0.05 g/L of manganese sulphate enhanced the antimicrobial activity of postbiotic RS5 by 108%. The cost of the production medium was reduced by 85% as compared to the commercially available de Man, Rogosa and Sharpe medium that is typically used for Lactobacillus cultivation. Hence, the refined medium has made the postbiotic RS5 more feasible and cost-effective to be adopted as a feed supplement for various livestock industries.
Fulltext Image reconstruction of a charge coupled device based optical tomographic instrumentation system for particle sizing

Idroas M, Rahim RA, Green RG, Ibrahim MN, Rahiman MH

Sensors (Basel), 2010;10(10):9512-28.
PMID: 22163423 DOI: 10.3390/s101009512

This research investigates the use of charge coupled device (abbreviated as CCD) linear image sensors in an optical tomographic instrumentation system used for sizing particles. The measurement system, consisting of four CCD linear image sensors are configured around an octagonal shaped flow pipe for a four projections system is explained. The four linear image sensors provide 2,048 pixel imaging with a pixel size of 14 micron × 14 micron, hence constituting a high-resolution system. Image reconstruction for a four-projection optical tomography system is also discussed, where a simple optical model is used to relate attenuation due to variations in optical density, [R], within the measurement section. Expressed in matrix form this represents the forward problem in tomography [S] [R] = [M]. In practice, measurements [M] are used to estimate the optical density distribution by solving the inverse problem [R] = [S](-1)[M]. Direct inversion of the sensitivity matrix, [S], is not possible and two approximations are considered and compared-the transpose and the pseudo inverse sensitivity matrices.
Fulltext Electrodynamics sensor for the image reconstruction process in an electrical charge tomography system

Rahmat MF, Isa MD, Rahim RA, Hussin TA

Sensors (Basel), 2009;9(12):10291-308.
PMID: 22303174 DOI: 10.3390/s91210291

Electrical charge tomography (EChT) is a non-invasive imaging technique that is aimed to reconstruct the image of materials being conveyed based on data measured by an electrodynamics sensor installed around the pipe. Image reconstruction in electrical charge tomography is vital and has not been widely studied before. Three methods have been introduced before, namely the linear back projection method, the filtered back projection method and the least square method. These methods normally face ill-posed problems and their solutions are unstable and inaccurate. In order to ensure the stability and accuracy, a special solution should be applied to obtain a meaningful image reconstruction result. In this paper, a new image reconstruction method - Least squares with regularization (LSR) will be introduced to reconstruct the image of material in a gravity mode conveyor pipeline for electrical charge tomography. Numerical analysis results based on simulation data indicated that this algorithm efficiently overcomes the numerical instability. The results show that the accuracy of the reconstruction images obtained using the proposed algorithm was enhanced and similar to the image captured by a CCD Camera. As a result, an efficient method for electrical charge tomography image reconstruction has been introduced.
Fulltext Multiple fan-beam optical tomography: modelling techniques

Rahim RA, Chen LL, San CK, Rahiman MH, Fea PJ

Sensors (Basel), 2009;9(11):8562-78.
PMID: 22291523 DOI: 10.3390/s91108562

This paper explains in detail the solution to the forward and inverse problem faced in this research. In the forward problem section, the projection geometry and the sensor modelling are discussed. The dimensions, distributions and arrangements of the optical fibre sensors are determined based on the real hardware constructed and these are explained in the projection geometry section. The general idea in sensor modelling is to simulate an artificial environment, but with similar system properties, to predict the actual sensor values for various flow models in the hardware system. The sensitivity maps produced from the solution of the forward problems are important in reconstructing the tomographic image.
Fulltext Ultrasensitive and Highly Selective Graphene-Based Field-Effect Transistor Biosensor for Anti-Diuretic Hormone Detection

Selvarajan RS, Rahim RA, Majlis BY, Gopinath SCB, Hamzah AA

Sensors (Basel), 2020 May 06;20(9).
PMID: 32384631 DOI: 10.3390/s20092642

Nephrogenic diabetes insipidus (NDI), which can be congenital or acquired, results from the failure of the kidney to respond to the anti-diuretic hormone (ADH). This will lead to excessive water loss from the body in the form of urine. The kidney, therefore, has a crucial role in maintaining water balance and it is vital to restore this function in an artificial kidney. Herein, an ultrasensitive and highly selective aptameric graphene-based field-effect transistor (GFET) sensor for ADH detection was developed by directly immobilizing ADH-specific aptamer on a surface-modified suspended graphene channel. This direct immobilization of aptamer on the graphene surface is an attempt to mimic the functionality of collecting tube V 2 receptors in the ADH biosensor. This aptamer was then used as a probe to capture ADH peptide at the sensing area which leads to changes in the concentration of charge carriers in the graphene channel. The biosensor shows a significant increment in the relative change of current ratio from 5.76 to 22.60 with the increase of ADH concentration ranging from 10 ag/mL to 1 pg/mL. The ADH biosensor thus exhibits a sensitivity of 50.00 µA· ( g / mL ) - 1 with a limit of detection as low as 3.55 ag/mL. In specificity analysis, the ADH biosensor demonstrated a higher current value which is 338.64 µA for ADH-spiked in phosphate-buffered saline (PBS) and 557.89 µA for ADH-spiked in human serum in comparison with other biomolecules tested. This experimental evidence shows that the ADH biosensor is ultrasensitive and highly selective towards ADH in PBS buffer and ADH-spiked in human serum.
Fulltext A New Method of Rice Moisture Content Determination Using Voxel Weighting-Based from Radio Tomography Images

Mohd Ramli NA, Fazalul Rahiman MH, Kamarudin LM, Mohamed L, Zakaria A, Ahmad A, et al.

Sensors (Basel), 2021 May 26;21(11).
PMID: 34073162 DOI: 10.3390/s21113686

This manuscript presents a new method to monitor and localize the moisture distribution in a rice silo based on tomography images. Because the rice grain is naturally hygroscopic, the stored grains' quality depends on their level of moisture content. Higher moisture content leads to fibre degradation, making the grains too frail and possibly milled. If the moisture is too low, the grains become brittle and are susceptible to higher breakage. At present, the single-point measurement method is unreliable because the moisture build-up inside the silo might be distributed unevenly. In addition, this method mostly applies gravimetric analysis, which is destructive. Thus, we proposed a radio tomographic imaging (RTI) system to address these problems. Four simulated phantom profiles at different percentages of moisture content were reconstructed using Newton's One-Step Error Reconstruction and Tikhonov Regularization algorithms. This simulation study utilized the relationship between the maximum voxel weighting of the reconstructed RTI image and the percentage of moisture content. The outcomes demonstrated promising results, in which the weighting voxel linearly increased with the percentage of moisture content, with a correlation coefficient higher than 0.95 was obtained. Therefore, the results support the possibility of using the RTI approach for monitoring and localizing the moisture distribution inside the rice silo.
Hardware Implementation of Multiple Fan Beam Projection Technique in Optical Fibre Process Tomography

Rahim RA, Rahiman MH, Chen LL, San CK, Fea PJ

Sensors (Basel), 2008 May 23;8(5):3406-3428.
PMID: 27879885

The main objective of this project is to implement the multiple fan beam projection technique using optical fibre sensors with the aim to achieve a high data acquisition rate. Multiple fan beam projection technique here is defined as allowing more than one emitter to transmit light at the same time using the switch-mode fan beam method. For the thirty-two pairs of sensors used, the 2-projection technique and 4- projection technique are being investigated. Sixteen sets of projections will complete one frame of light emission for the 2-projection technique while eight sets of projection will complete one frame of light emission for the 4-projection technique. In order to facilitate data acquisition process, PIC microcontroller and the sample and hold circuit are being used. This paper summarizes the hardware configuration and design for this project.
An Optical Tomography System Using a Digital Signal Processor

Rahim RA, Thiam CK, Rahiman MH

Sensors (Basel), 2008 Mar 27;8(4):2082-2103.
PMID: 27879811

The use of a personal computer together with a Data Acquisition System (DAQ) as the processing tool in optical tomography systems has been the norm ever since the beginning of process tomography. However, advancements in silicon fabrication technology allow nowadays the fabrication of powerful Digital Signal Processors (DSP) at a reasonable cost. This allows this technology to be used in an optical tomography system since data acquisition and processing can be performed within the DSP. Thus, the dependency on a personal computer and a DAQ to sample and process the external signals can be reduced or even eliminated. The DSP system was customized to control the data acquisition process of 16x16 optical sensor array, arranged in parallel beam projection. The data collected was used to reconstruct the cross sectional image of the pipeline conveyor. For image display purposes, the reconstructed image was sent to a personal computer via serial communication. This allows the use of a laptop to display the tomogram image besides performing any other offline analysis.
Fulltext Current and Potential Developments of Cortisol Aptasensing towards Point-of-Care Diagnostics (POTC)

Zainol Abidin AS, Rahim RA, Md Arshad MK, Fatin Nabilah MF, Voon CH, Tang TH, et al.

Sensors (Basel), 2017 May 22;17(5).
PMID: 28531146 DOI: 10.3390/s17051180

Anxiety is a psychological problem that often emerges during the normal course of human life. The detection of anxiety often involves a physical exam and a self-reporting questionnaire. However, these approaches have limitations, as the data might lack reliability and consistency upon application to the same population over time. Furthermore, there might be varying understanding and interpretations of the particular question by the participant, which necessitating the approach of using biomarker-based measurement for stress diagnosis. The most prominent biomarker related to stress, hormone cortisol, plays a key role in the fight-or-flight situation, alters the immune response, and suppresses the digestive and the reproductive systems. We have taken the endeavour to review the available aptamer-based biosensor (aptasensor) for cortisol detection. The potential point-of-care diagnostic strategies that could be harnessed for the aptasensing of cortisol were also envisaged.
Detection of Citrus tristeza virus by using fluorescence resonance energy transfer-based biosensor

Shojaei TR, Salleh MA, Sijam K, Rahim RA, Mohsenifar A, Safarnejad R, et al.

Spectrochim Acta A Mol Biomol Spectrosc, 2016 Dec 05;169:216-22.
PMID: 27380305 DOI: 10.1016/j.saa.2016.06.052

Due to the low titer or uneven distribution of Citrus tristeza virus (CTV) in field samples, detection of CTV by using conventional detection techniques may be difficult. Therefore, in the present work, the cadmium-telluride quantum dots (QDs) was conjugated with a specific antibody against coat protein (CP) of CTV, and the CP were immobilized on the surface of gold nanoparticles (AuNPs) to develop a specific and sensitive fluorescence resonance energy transfer (FRET)-based nanobiosensor for detecting CTV. The maximum FRET efficiency for the developed nano-biosensor was observed at 60% in AuNPs-CP/QDs-Ab ratio of 1:8.5. The designed system showed higher sensitivity and specificity over enzyme linked immunosorbent assay (ELISA) with a limit of detection of 0.13μgmL(-1) and 93% and 94% sensitivity and specificity, respectively. As designed sensor is rapid, sensitive, specific and efficient in detecting CTV, this could be envisioned for diagnostic applications, surveillance and plant certification program.
Detection of Vancomycin-Resistant Enterococcus Spp. (VRE) from Poultry

Toosa H, Radu S, Rusul G, Latif AR, Rahim RA, Ahmad N, et al.

Malays J Med Sci, 2001 Jan;8(1):53-8.
PMID: 22973157

Twenty-eight isolates of E. faecalis and 5 isolates of E. hirae were isolated from chicken samples obtained from markets in Sri Serdang, Selangor. They were tested for susceptibility to vancomycin and other antimicrobial agents. All of the isolates showed multiple resistance to the antibiotic tested. All Enterococcus spp. were resistant (100%) to ceftaxidime, cephalothin, erythromycin, gentamicin, kanamycin, nalidixic acid and streptomycin. Resistance was also observed to norfloxacin (97%), tetracycline (91%), penicillin (85%), bacitracin (82%), chloramphenicol (61%) and the least resistance was to ampicillin (27%). High prevalence to vancomycin resistance was detected among the E. faecalis (27of 28) and E. hirae (4 of 5) isolates. The multiple antibiotic resistance index ranging between 0.64 to 1.0 showed that all strains tested originated from high-risk contamination. Plasmid profile analysis of Enterococcus spp. revealed plasmid DNA bands ranging in size from 1.3 to 35.8 megadalton but some isolates were plasmidless. No correlation could be made between plasmid patterns and antibiotic resistance.
CD47 and CD36 expressions in the placenta of mothers with chorioamnionitis

Hanim BS, Rahim RA, Mohd Salleh MF, Zakaria H, Abd Shukor N

Malays J Pathol, 2023 Dec;45(3):463-471.
PMID: 38155387

INTRODUCTION: Chorioamnionitis is the inflammation of the placenta and is histologically defined as the presence of neutrophilic infiltration into the chorio-amnion membrane with and without involvement of the umbilical cord. Currently, the inflammatory mediators involved in the eliciting of inflammatory response is still largely under investigation. CD47 and CD36 are pro-inflammatory molecules that are still under investigation. The aim of this study was to determine the expressions of CD47 and CD36 in the placenta of mothers with chorioamnionitis.
MATERIALS AND METHODS: This was a cross-sectional study, involving a total of 100 cases that comprised of acute subchorionitis (stage I, n=20), acute chorioamnionitis (stage II, n=20), acute necrotising chorioamnionitis (stage III, n=20) and non-chorioamnionitis placenta as control (n=40). All tissue blocks were retrieved from the archived pathology record over a period of 4 years. CD36 and CD47 immunohistochemistry were performed on all cases and their expression in various cell types on the placenta were analysed.
RESULTS: CD36 was expressed only on the foetal vascular endothelial cells. Interestingly, CD47 showed positive staining on the neutrophils and its expression was significantly different between maternal inflammatory response stage II chorioamnionitis (n=13/20, p<0.001) with stage I and stage III chorioamnionitis.
DISCUSSION: Our study showed CD47 was expressed in the neutrophils and it was associated with poorer perinatal outcomes and it may have a role in the pathogenesis of chorioamnionitis.
Characterization of vibrio parahaemolyticus isolated from coastal seawater in peninsular Malaysia

Tanil GB, Radu S, Nishibuchi M, Rahim RA, Napis S, Maurice L, et al.

Southeast Asian J. Trop. Med. Public Health, 2005 Jul;36(4):940-5.
PMID: 16295549

Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.
Prevalence of Listeria spp and Listeria monocytogenes in meat and fermented fish in Malaysia

Hassan Z, Purwati E, Radu S, Rahim RA, Rusul G

Southeast Asian J. Trop. Med. Public Health, 2001 Jun;32(2):402-7.
PMID: 11556596

Fermented fish and meat samples were purchased from supermarket and wet market for microbiological analysis of Listeria species and Listeria monocytogenes contamination. Listeria species were isolated from 17 (73.9%) of 23 samples of imported frozen beef, 10 (43.5%) of the 23 samples of local beef and 14 (56%) of the 25 samples of fermented fish from wet market. Listeria monocytogenes occurred in 15 (75%) of the frozen beef samples, 6 (30.4%) of the 23 samples of local meat and 3 (12%) of the 25 samples from fermented fish. Listeria species was not isolated from any of the 23 samples of imported frozen beef from supermarket and from the 5 samples of buffalo meat examined. This highlights the possibility of Listeria spp or L. monocytogenes to persist in meat and fermented fish in wet market and raises the problem of illness due to the handling and consumption of Listeria-contaminated meat or fermented fish are likely as evidence by the high contamination rates of samples sold at the wet market.
Detection and molecular characterization of the zot gene in Vibrio cholerae and V. alginolyticus isolates

Raja N, Shamsudin MN, Somarny W, Rosli R, Rahim RA, Radu S

Southeast Asian J. Trop. Med. Public Health, 2001 Mar;32(1):100-4.
PMID: 11485069

A total of 11 Vibrio cholerae isolates from 1996-1998 outbreaks in Malaysia and 4 V. alginolyticus were analyzed. Isolates were characterized by polymerase chain reaction (PCR) and Southern hybridization for the presence of the gene encoding zonula occludens toxin (zot). Screening of zot gene by PCR revealed the presence of this gene in V. cholerae and V. alginolyticus. The zot gene from one V. cholerae Ogawa isolate that was cloned in a pCR 2.1 TOPO vector was sequenced. The sequences obtained were 99% homologous to the zot gene sequence from the Gene Bank.
Fulltext Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis

Yap TW, Rabu A, Abu Bakar FD, Rahim RA, Mahadi NM, Illias RM, et al.

ScientificWorldJournal, 2014;2014:642891.
PMID: 24982972 DOI: 10.1155/2014/642891

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.
Catharanthus roseus Aqueous Extract is Cytotoxic to Jurkat Leukaemic T-cells but Induces the Proliferation of Normal Peripheral Blood Mononuclear Cells

Ahmad NH, Rahim RA, Mat I

Trop Life Sci Res, 2010 Dec;21(2):101-13.
PMID: 24575203

Research on natural products has been widely used as a strategy to discover new drugs with potential for applications in complementary medicines because they have fewer side effects than conventional drugs. The aim of the present study was to evaluate the in vitro cytotoxic effects of crude aqueous Catharanthus roseus extract on Jurkat cells and normal peripheral blood mononuclear cells (PBMCs). The aqueous extract was standardised to vinblastine by high performance liquid chromatography (HPLC) and was used to determine cytotoxicity by the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. DNA fragmentation assay was employed to determine if cell death was due to apoptosis. The results showed that the aqueous extract induced cell death of Jurkat cells at 24, 48 and 72 hours post-treatment in a time- and dose-dependent manner. However, cells treated at 48 and 72 hours produced higher cytotoxic effects with half maximal inhibitory concentration (IC50) values of 2.55 μg/ml and 2.38 μg/ml, respectively. In contrast, the extract induced normal PBMC proliferation, especially after 24 hours treatment with 1000 μg/ml. This result indicates that the C. roseus crude aqueous extract showed differential effects of inhibiting the proliferation of the Jurkat cell line and promoting the growth of PBMCs. These data suggest that the extract may be applicable for modulating the normal and transformed immune cells in leukaemia patients.
Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity

Zamri HF, Shamsudin MN, Rahim RA, Neela V

Vaccine, 2012 May 2;30(21):3231-8.
PMID: 22426330 DOI: 10.1016/j.vaccine.2012.02.012

A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 10(9) cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.
Fulltext K-Ras Peptide Mimotope Induces Antigen Specific Th1 and B-Cell Immune Responses against G12A-Mutated K-Ras Antigen in Balb/c Mice

Siak PY, Wong KY, Song AA, Rahim RA, In LLA

Vaccines (Basel), 2021 Feb 26;9(3).
PMID: 33652552 DOI: 10.3390/vaccines9030195

KRAS G12A somatic point mutation in adenocarcinomas is categorized clinically as ineligibility criteria for anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies. In this study, a modified G12A-K-ras epitope (139A) with sequence-specific modifications to improve immunogenicity was developed as a potential vaccine against G12A-mutant KRAS cancers. Additionally, coupling of the 139A epitope with a tetanus toxoid (TTD) universal T-cell epitope to improve antigenicity was also reported. To facilitate convenient oral administration, Lactococcus lactis, which possesses innate immunomodulatory properties, was chosen as a live gastrointestinal delivery vehicle. Recombinant L. lactis strains secreting a G12A mutated K-ras control and 139A with and without TTD fusion were generated for comparative immunogenicity assessment. BALB/c mice were immunized orally, and high survivability of L. lactis passage through the gastrointestinal tract was observed. Elevations in B-cell count with a concomitant titre of antigen-specific IgG and interferon-γ secreting T-cells were observed in the 139A treated mice group. Interestingly, an even higher antigen-specific IgA response and interferon-γ secreting T-cell counts were observed in 139A-TTD mice group upon re-stimulation with the G12A mutated K-ras antigen. Collectively, these results indicated that an antigen-specific immune response was successfully stimulated by 139A-TTD vaccine, and a TTD fusion was successful in further enhancing the immune responses.

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