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*Corresponding author: E-mail: ibrahimshnawa3@gmail.com; 
 
Cite as: IMS, Shnawa, Algerbori HSH, and Thewaini QNO. 2024. “Immunogenicity of Bacterial Protoplasmic Sonicate 
Proteins”. Asian Journal of Immunology 7 (1):175-80. https://journalaji.com/index.php/AJI/article/view/141. 
 
 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 175-180, 2024; Article no.AJI.123078 
 

 
 

 

 

Immunogenicity of Bacterial 
Protoplasmic Sonicate Proteins 

 
Shnawa IMS a,b*, Algerbori HSH c and Thewaini QNO a 

 
a Department of Medical Biotechnology, College of Biotechnology, AL-Qasim Green University, 

Qasim, Babylon Province, Iraq. 
b College of Nursing Technology, University of Hilla, Babylon Province, Iraq. 

c Department of Basic Sciences, College of Dentistry, University of Babylon, Babylon Provine, Iraq. 
 

Authors’ contributions  
 

This work was carried out in collaboration among all authors. All authors read and approved the final 
manuscript. 

 

Article Information 
 

DOI: https://doi.org/10.9734/aji/2024/v7i1141  
 

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Received: 04/07/2024 
Accepted: 06/09/2024 
Published: 20/09/2024 

 
 

ABSTRACT 
 

Bacterial Protoplasmic Sonicate Proteins BPSP were recovered from skin burn Pseudomonas 
aeruginosa and Klebsiella oxytoca infections. These proteins   were considerd as immunogens in 
rabbits. Mucosal and systemic, humoral and cellular immune immune responses were matched. 
The P. aeruginosa protoplasmic sonicate proteins induces an increase in; NBT % neutorphil 
phagocytosis, significant leukocyte migration inhibition cytokines, IL6 cytokine elevation and rise up 
of humoral agglutinins responses. While, K. oxytoca protoplasmic sonicate proteins induces 
increase of; humoral agglutinins and IL6 cytokine as well as non-signifcant increase in leukocyte 
inhibitory cytokines. Both at mucosal and systemic compartments. The study highlights the 
potentials of these proteins as candidates for developing vaccines against multidrug resistance 
infections, particularly in burn patients. These findings underscore the importance of 
immunogenicity in advancing bacterin development in clinical use. 

 

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1141
https://www.sdiarticle5.com/review-history/123078


 
 
 
 

Shnawa et al.; Asian J. Immunol., vol. 7, no. 1, pp. 175-180, 2024; Article no.AJI.123078 
 
 

 
176 

 

Keywords: Antigens; bacterin; cellular; humoral; immunogen; protein; vaccine. 
 

1. INTRODUCTION 
 
Bacterial antigens [BAGs] were being 
investigated to be of use in, bacterial diagnosis, 
bacterial infection diagnosis, vaccine   
development and vaccine production. 
Immunogenicity of BAGs holds a backbone 
position in vaccine development strategies                      
[1–7]. The objective of the present work                     
was the investigation of immunogenicity of 
protoplasmic sonicate protein antigens of skin 
burn infections with P. aeruginosa and                         
K. oxytoca. 
 

2. MATERIALS AND METHODS 
 

2.1 Protoplasmic Sonicate Protein 
Antigens P SPA 

 
The PSPA separation was in direct way for P. 
aeruginosa [8], while, by in direct way as it needs 
removal of the capsule [9,10] before processing 
for obtaining PSPs. The method for separation, 
identification, purification and quantitation of 
these PSPs were as in the method described by 
Bjorn et al. [8]. The details of the processing 
method was  as in the followings; Six mls of tris 
buffer 0.01 N and PH 8 was added to the surface 
of  24 hrs growth of P. aeruginosa and K. 
oxytoca (after suspending bacteria then removal  
of capsule [9,10]) on the nutrient agar plates. 
Growth were   vortoxed in vortox test tubes for 
thee minutes. Suspensions were centrifuged at 
5000 rpm for ten minutes. Supernatant were 
discarded and  pellets Ps  were   kept. Ps  
suspensions were tubbed and washed three 
times with  tris buffer 0.01N.Ps were 
reconstituted with 6 mls, and were  tubbed in the  
cell disintegrator tubes. Then jacketed with 
cooled ice. The best sonication conditions were 
five times for five minutes at 20 oscillation 
ampiltude. The sonicated cell suspensions were 
centrifuged at 5000 rpm for ten minutes. 
Supernats were collected and ultrafiltred with 
0.22 um millipore filter. Fiterates were collected 
in sterile plastic tubes, then proteins were 
separated with PEG 6000 ,6% as inShnawa and 
AlSadi [11]. The protein concentration 
determination was measured by Biurt test [12]. 
To this end the preparations can be designated  
as PAPSP and KOPSP both P. aeruginosa                   
and K.oxytoca respectively. These PSPs                   
were distributed into alliqoutes of 0.5 mls.                 
In an appendroff plastic tubes and kept at -
20C,till use. 

2.2 Immune Reagents 
 
Specific immune priming of rabbits were done 
with the PSP concentrations of 2.71 mg/l for 
PAPSP and 1.8 md/l for KOPSP [12]. The 
somatic antigens for both bacteria were prepared 
as heat killed  as  in [13].Complete Freund 
Ajuvant  that from Difco, Co.Ltd. The test 
proposed immunogens    were made as; one 
volume of CFA mixed with one volume the test 
proteins Immunization Protocols 
 

2.3 Immunization Protocols 
 
A mounthwise twice dosage of PSP-CFA in 2 mls 
amounts were dosaged in the first and second 
months to the rabbits. The specific immune 
priming was that of multisite injection protocol 
[14]. 
 

2.4 Rabbits 
 

A local breed rabbits brought from the local 
market were checked for ecto and 
endoparasites. As well as for pyrogens and 
found to be free off. They were acclimatized for 
housing conditions two weeks befor 
experimentation at and libitum conditions. Among 
which nine were elected and subdivided into two 
test and one control group each of three rabbits. 
 

2.5 Samplings and Immune Function 
Tests 

 

At the temination of the specific immune priming 
protocols, blood with and with out heparin were 
collected from the test and control rabbits by 
cardiac puncture for humoral and cellular 
immune tests. Sera were saved for serology and 
cytokine studies. Heparinized blood were used 
for leukocyte inhibitory factor [15] and for NBT 
phagocytosis [16]. Appendix for test and control 
rabbits were collected and open up, washed from 
digesta and processed for separation of mucosal 
globulins [17]. Mucosal leukocytes were 
separated by dextran 2% as in [18]. IL 6 
determinations were made as in the 
recommendation of the instruction of the 
manufacturer. Standared tube agglutination test 
were made as in [19]. 
 

3. RESULTS 
 

The NBT neutrophil phagocytosis percentages in 
PAPSPA (56.75 % for mucosal and36.5 for 



 
 
 
 

Shnawa et al.; Asian J. Immunol., vol. 7, no. 1, pp. 175-180, 2024; Article no.AJI.123078 
 
 

 
177 

 

systemic) and KOPSPA (48.5% for mucosal, 
41% for systemic) primed rabbits were higher 
than that of control rabbits (20% for 
mucosal,18% for the systemic). Leukocyte 
inhibitory cytoknine LIF % in PAPSP primed 
rabbits were  56.6% for mucosal and 58.45  for 
systemic as compared to control ,the mucosal 
was 90% and systemic was 86%.While for 
KOPSPA primed rabbits were; 88% for mucosal 
and 87.5% for systemic  LIF as compared to 
normal control were  93% for mucosal and 89% 
for systemic responses. The IL6 concentration 
determinations were shownig that  PAPSPA and 
KOPSPA primed rabbits have got higher IL6 
concentration  means  than normal control 
rabbits. PAPSPA primed rabbits IL6 
concentration means were 92.8 for mucosal and 
72.7 for systemic responses. In KOPSPA primed 
rabbits the mucosal concentration means were 
83.26 and for systemic were 76.79 as compared 
to normal control  were 8.83 for mucosal  and 
9.25 for systemic responses. The humoral 
specific agglutinin titre levels for PAPSPA were 
128 for mucosal and 320 for the systemic 
responses and KOPSP primed rabbits were 64 
for mucosal and 640 for systemic responses as 
compared to control rabbits were 4 for mucosal 
and 20 for systemic responses. Mucosal 
agglutinins were resistant to treatment with 2ME, 
Tables 1 and 2.  
 

4. DISSCUSSION 
 
The concept, application and continuity of the 
immunogenicity theme for bacterial protein 

antigens are still in the current mode of 
researchers allover the world [1-7]. 
Immunogenicity appeared to have two main 
facets.First that of theoritical immunologisets 
which advocate that immunogenicity is denoted 
to self-nonself recognition theme [6]. While the 
second facets was that for most of the proper 
immunologists which can be summerized as the 
ability of an antigen to initiate humoral and/or 
cellular conversion from the normal baseline 
immune functions to an optimized cellular 
immune reactions outcomes that are finalized by 
the optimest synthesis and production of              
cellular secretory proteins (antibodies, cytokines) 
concentrations and /or optimised depression of 
such secretory proteins [1-5,7]. Immunogenicity 
appeared to be essential for diagnosis and 
prophylaction of human infections as well as in 
cancer personalized prophylactic and therpeutic 
medicine. Hence the presnt study for 
Pseudomonas and Klesiella immunogenicity may 
participate in developing of a porototype bacterial 
protein based vaccine and/ or in development for 
an autovaccine for multidrug resistant skin burn 
infection [20]. 
 
The antigenic make up of bacteria                               
[1-5,7] like that of P. aeruginosa are                
formed from; flagella, pili, exottoxin A, 
exoppolysaachride, LPS, OMP, hemolysin, 
elastases, proteasis, heat stable phospholipid, 
heat stable glycolipid, exoenzyme S and 
ribosome [21-23]. While that of Klebsiella 
oxytoca are; capsule, somatic antigens and LPS 
[24]. Hence, PAPSP and KOPSP protein 

 
Table 1. The immunogenicity of PAPSP in primed rabbits and controls 

 
Rabbits groups NBT% LIF% IL6 pg/ml. Agglutinin titres 

PAPSPA     

M 
S 

46.75 
36.6 

56.6 
58.45 

2.8 
72.7 

128 
320 

Control     

M 20 93 92.8 4 
S 18 89 72.7 20 

 
Table 2. The immunogenicity of KOPSP in primed and control rabbits 

 
Rabbit groups NBT% LIF % IL6 pg/ml Agglutinin titres  

KOPSPA      

M 
S 

48.5 
41 

88 
87 

48 
72 

64 
640 

 

Controls      

M 
S 

20 
18 

93 
89 

8.83 
9.25 

4 
20 

 



 
 
 
 

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178 

 

Table 3. The immune features of the study bacterial proteins 
 

Features PAPSP [25] KOPSP [25] 

1-Chemical nature 
2-Origin 
3-Location 
4-Processing method 
5-Non-specific immune function 
 
6- Specific cellular immune 
function 
7-Induction of cytokine network 
8-Humoral Immune responses 
 
9-Functional Epitope Mapping 
[26,27] 
 
10-Immuen system 
compartments 
 
11-Expected Immune 
potentials [20] 

Protein 
Bacterial 
Intracellular 
Ultrasonication 
Rise up of phagocytosis By 
NBT% 
Significant inhibition of LIF 
cytokine 
Rise up of IL6 cytokine 
Rise up of specific agglutinins 
 
T cell dependent and T cell in-
ndepedent epitopes 
 
Mucosal and Systemic 
 
 
Prototype protein based 
vaccine and an autovaccine 

Protein 
Bacterial 
Intracellular 
Ultrasonication 
Rise up of phagocytosis by 
NBT % 
Nonsignificant inhibition of LIF 
cytokines 
 
Rise up of IL6 cytokines 
 
Rise up of specific agglutinins 
T cell independent and /or, 
TH2 dependent B cell epitope 
Mucosal and systemic 
 
 
Prototype protein based 
vaccine and an autovaccine. 

 
antigens are novel antigens prepared in this 
experimental settings. 
 

PAPS was proved to be immunogenic in rabbits 
model, Table 1, promoting humoral and cellular 
immune responses both at mucosal and 
systemic copartments. These findings were in 
contradiction with that of [24]. While, KOPSP 
immunogenicity, Table 2, were by promoting 
humoral immune responses both at mucosal and 
systemic level [21]. Different burn infecting 
bacterial protein preparations have shown 
different immune potential features, Table 3,[25]. 
The functional epitope mapping for PAPSP may 
be T cell depndent and T cell independent, while 
that of KOPSP may be of T cell independent or 
Th2 cell depent B cell epitopes [26,27]. Since 
immunogenicity is an integral part for developing 
bacterins. Such findings holds the position of 
novelity, based upon the need for an autogenous 
bacterins for multidrug resistant skin burn 
bacterial infections [28,29]. 
 

5. CONCLUSIONS 
 

P. aeruginosa PAPSP and K. oxytoca KOPSP 
protoplasmic sonicate proteins were found as 
lapin immunogens. PAPSP mediate humoral and 
cellular immune responses both at mucosal and 
systemic compartments. While, KOPSP mediate 
humoral immune responses both at mucosa and 
blood stream. These proteins may be of 
expected immune potentials as protein based 
vaccine candidate and as an autovaccine for 
multidrug resistant skin burn infections. 

DISCLAIMER  

 
Authors hereby declare that NO generative AI 
technologies such as Large Language Models 
(ChatGPT, COPILO, etc) and text-to-image 
generations have been used during writing or 
editing of this manuscript.  

 
CONSENT 
 
It is not applicable. 
 
 

ETHICAL APPROVAL 
 

The housing, handling and interventions on 
rabbits were done following the international acts 
for regulation of housing, handling and 
interventions. 

 
COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 
 

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