







































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: ibrahimshnawa3@gmail.com; 
 
Cite as: Shnawa, Ibrahim M S, Azhar O AL-Thahab, and Kasim N A.Thewaini. 2025. “Preclinical Immunogenicity of Capsule 
and Outermembrane Proteins of a Local Neurotropic Isolate of Hemophilus Infleunzae B in a Lapin Model”. Asian Journal of 
Immunology 8 (1):1-7. https://doi.org/10.9734/aji/2025/v8i1155. 
 

 
 

Asian Journal of Immunology 
 
Volume 8, Issue 1, Page 1-7, 2025; Article no.AJI.129951 
 

 
 

 

 

Preclinical Immunogenicity of Capsule 
and Outermembrane Proteins of a 

Local Neurotropic Isolate of 
Hemophilus Infleunzae B in a Lapin 

Model 
 

Ibrahim M S Shnawa a,b*, Azhar O AL-Thahab c  
and Kasim N A.Thewaini a 

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

Babylon, Iraq. 
b College of Nursing, University of Hillah, Babylon, Iraq. 

c Department of Biology, College of Science, University of Babylon, Babylon, 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/2025/v8i1155  

 
Open Peer Review History: 

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer 
review comments, different versions of the manuscript, comments of the editors, etc are available here: 

https://pr.sdiarticle5.com/review-history/129951  

 
 

Received: 26/11/2024 
Published: 06/02/2025 

 
 

ABSTRACT 
 

The preclinical immunogenicity of capsule C and outer membrane protein OMP of human 
neurotropic isolate of Hemophilus influenzae b HIB in a lapin model is being reported. Hemophilus 
influenzae b is gram negative, short rods, encapsulated bacterium, microaerophilic, fastidious, need 

Original Research Article 

https://doi.org/10.9734/aji/2025/v8i1155
https://pr.sdiarticle5.com/review-history/129951


 
 
 
 

Shnawa et al.; Asian J. Immunol., vol. 8, no. 1, pp. 1-7, 2025; Article no.AJI.129951 
 
 

 
2 
 

X and V factor for growth with six capsular serotypes. Among which serotype b was the dominant. 
Itis associated with human pyogenic respiratory and meningeal infections. The C and OMP were 
separated, purified, and characterized. Then, quantfied to the rate of 3 mg/m L and 2.27 mg/mL 
accordingly. Both of the preparations were used as test antigens for the invitro use and for speific 
immune priming of rabbits. Cell free culture filtrate of test isolate was prepared and used as a skin 
sensitin in DTH test. Rabbits were were preconditioned with CFA adjuvant for two weeks then 
specific immune primed using multisite multi-injection protocols. Capsule immunogen induce 
leukocyte stimulatory factor cytokines. Four to six folds rise both titres and concentrations of anti 
capsular precipitins and anti OMP hemagglutinins were noted. Both C and AMP are proved to be 
lapin immunogens stimulate humoral immune responses at both systemic and mucosal immune 
compartments. These findings may forms the background information and be an integral part of 
preclinical and clinical development and production of local H. infleunzae b systemic and/or 
mucosal vaccines. Which could be valid on approval for local mass vaccination against childhood 
meningitis in this area. 
 

 
Keywords: Antigen; cellular; humoral; immunogens; vaccines. 
 

1. INTRODUCTION 
  
Hemophilus influenzae b is short gram negative 
rods, encapsulated bacterium. Microaerophilic, 
fastidious need X and V factor for growth with six 
capsular serotypes. Among which serotype b 
was the dominant. Hib is associated with human 
pyogenic respiratory and meningeal infections 
(Levenison et al., 2018). Meningitis is a life 
threatining disease affects millions of people of 
all ages allover the world and is considered a 
major global public health issue (International 
Society of Infectious Diseases 2023). The nature 
of the immunity induced by H. Infleunzae b in 
rabbit, rat and mice is humoral by a T cell 
independent epitopes (Levenison et al., 2018; 
Wisplwey et al., 1989). Circulating anti capsular 
PRP antibodies promote complement dependent 
bactericidal power and phagocytosis found in 
meningitis patients and vaccinee. There is a 
correlation between the presence of bactericidal 
antibodies and resistance to H. infleunzae b 
infection (International Society of Infectious 
Diseases 2023; Wisplwey et al., 1989). 
Hemophilus infleunzae b OMP have been proved 
to be immunogens in infant rat (Hansen et al., 
1981). The noncapsulated atypical H. infleunzae 
suppress and modulate cellular and humoral 
immune responses to atypical H. Infleunzae 
vaccine due to interference phenomena (Mawas 
et al., 2006; Lee et al., 2010; Griffiths et al., 
2012; Latez et al., 2004; Santosham, 1993). 
OMP of atypical H. infleunzae is affected by                    
the function of Treg on B cells specific for                   
OMP in a cell culture system (Hirano et al., 
2019). The objective of the present work was to 
map immunogenicity of capsule and outer 
membrane proteins of H. infleunzae b local 
isolate. 

2. MATERIALS AND METHODS 
 

2.1 Reviewer Inquiry 
  

It is a reviva revista study that has been a part of 
AL Thahab Ph. D. Thesis at 2006 on HIB 
meningitis; pathogenesis and immunology in 
man and laboratory animal. Prof. Shnawa and 
Ass. Prof. Thewaini had been supervisor and 
coadvisor. Whereby had been faculty 
membership at Babylon University. 
 

2.2 Neuropathic H. Infleunzae 
 

Local H. infleunzae b isolate from a clinically 
proven human meningitis cases The isolate was, 
culturally and biochemically characterized 
through classical and API20E system. Serotyped 
using Difco, Co, BDTM kit (Gonzalez & Ledeboer 
2023). 
  

2.3 Preparation of Immune Reagents 
  

The capsule was saparated, purified, identified 
and quantified to the concentration of 3 mg/ml 
(Kwapnski, 1972). OMP was separated, purified, 
identified and quantified to the concentration of 
2.27 mg/ml (Catlin et al., 1972). Sensitin was 
prepared as cell free culture filtrates as in 
(Shnawa & Thewaini 2002). 
 

2.4 Rabbits 
  
A Newzeland white rabbits weighing 1- 1.5 kgs 
were checked to be free of parasite, bacteria, 
and antibodies. These rabbits were adapted to 
housing conditions for one week and kept ad 
libitum conditions. They were grouped and 
assigned into three groups each of three. Two 
test groups and one control group. 



 
 
 
 

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3 
 

2.5 Immunization Protocol 
  

The test groups were primed with complete 
Freund adjuvant CFA in a rate of one ml for each 
rabbit, control group was primed with saline. Test 
and control groups were left for two weeks. After 
preconditioning with CFA. One group was primed 
with capsule, 3 mg/L. and the other with OMP 
2.27 mg/mL. in a two weeks apart multisite multi-
injection protocol followed by one weeek leave 
then test bleed (AlShahery & Shnawa 1989). 

 

Capsule in 3 mg/L...................... three rabbits 
Outermemberane protein 2.27 
mg/L.....................three rabbits 
Saline sham control....................three rabbits 
 

2.6 Sampling and Processing 
  

Blood samples were collected from test and 
control rabbits by cardiac punctutre with a rate of 
six mls from each rabbit. Samples were divided 
into two parts each of three mls amounts. Tubes 
without anticoaggulant and three mls., and tube 
with anticoaggulant. Sera were saved from 
samples without anticoaggulants in a rate of 0.5 
ml amounts in an append roff tubes and kept at -
18 C. Whole blood with anticoaggulant uesed for 
test for leukocyte inhibitory factors by capillary 
method (Soberg, 1969). Appendecies were 
collected from primed and control rabbits. They 
were opened up, freed from digesta and 
scrapped with sterile clean scalpel to remove 
mucus from the mucosal surfaces. Proportional 
mucus-saline were mixed in a sterile petri-dish. 
Mixtures were tubbed in centrifuge tubes and 
centrifuged at 5000 rpm for ten minutes. 
Supenates of three mls amounts were mixed with 
equal amounts of PEB 6%, 6000 and left at 4 C 
for one hr and centrfuged for 5000 rpm for 15 
minutes. Precipitates were saved and 
reconstituted with sterile formal normal saline 
(Shnawa & Thewaini 2002). 
 

2.7 Immune Function Tests 
  

The anticapsular antisera and mucosal globulin 
were titrated with capsule antigen using capillary 
tube precipitation test (Turgeon, 2020). The anti 
OMP antisera and mucosal globulin were titrated 
with OMP coated tanned sheep red cells by 
micro hemagglutination test (Joshi & Chauhan 
2022). Mucus-Saline mixture was mixed with 
dextran solutions in an equal amounts for 
separation of mucosal leukocytes as in Metcalf et 
al. (1986). Systemic and mucosal leukocyte 
inhibitory factor was done by capillary method as 
in Soberg (1969).  

3. RESULTS 
 

3.1 Baseline Immune Functions 
  
Control rabbits reveals the normal baseline 
immune functions in this experimental settings. 
The serum globulin protein concentration was 
6.75 g/L. While, the normal appendex glubulin 
protein concentration was 0.425 g/L. Natural 
serum baseline titre mean was 10 both for anti-
capsular and anti-OMP antibodies and normal 
mucosal globulin concentration were 0.425 and 
0.4g/L. both for anti-capsular and anti-OMP 
antibodies. While the normal leukocyte inhibitory 
factor cytokines were ranging between 0.95 and 
0.97%. 
  

3.2 Cellular Immune Conversion 
  
Specific OMP priming to rabbits lead to non-
significant leukocyte inhibitory factor cytokines 
and mild leukocyte stimulatory factor cytokines in 
one rabbit replicate both at mucosal and 
systemic responses. Specific capsular primed 
rabbits have shown leukocyte stimulatory factor 
cytokines as compared to LIF of normal baseline 
immune functions. OMP and Capsular immune 
primied rabbits indicate cellular immune 
conversion than that of basline cellular immune 
functions, Table 1. 
 

3.3 Delayed Allergenic Immune 
Conversion 

  
AT 72 hrs post-sensitization with the skin 
sensitins CFCF through intradermal injection of 
the primed rabbits. Neither classical DTH nor 
Jone-Moote reaction noted in both of the primed 
groups. This may indicate that there were no 
delayed allergenic immune conversion in these 
immune primed test rabbits Table 1. 
 

3.4 Humoral Immune Conversion 
  
The mean of serum anticapsular antibody titre 
was 533.3 compared to the baseline humoral 
immune function was 10. While, the mucosal 
anti-capsular antibody mean titre was 42.66. As 
compare to normal humoral baseline immune 
function was 2. In other word five folds increase 
folds increase inthe serum titer means and four 
folds in the mucosa mean titres. While, the mean 
serum anti-OMP titre was 853.3 compared to 
normal baseline function mean titre was 10. The 
mucosal anti-OMP mean titre was 85.33 
compared to normal baseline humoral immune 
fuction was 2. That is to say six folds increase in 



 
 
 
 

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4 
 

serum and five folds im mucosal anti-OMP 
antibodies. The folds increase in mean antibody 
titres in serum and in mucosa indicated humoral 
immune conversions, Table 2. 
 

4. DISCUSSION 
  
The OMP and capsular polysaccharide of H. 
influenzae b are; pathogenicity determnant, 
virulence associated antigens and virulence 
factor. So both in conjugate state or in separate 
state they are standing as targets for vaccine 
candidates and human approved vaccines in 
more than one vaccine qualifying boards all over 
the world (Shnawa, 2019). In the present 
communication tempts to investigate 
immunogenicity of subcellular fractions of local 
neurotropic neurogenic H. infleunzae b. These 
subfractions are; Capsule and OMP in a lapin 
model, Tables 1 &2. The specific immune 
priming protocols tempts to precondition rabbits 
with Freund Complete Adjuvant CFA for a period 
of two weeks that provok non-specific immune 
stimulation to both of innate and adaptive 
immune cells (AlShahery & Shnawa 1989). As a 
pre-immunization adjuvant followed by two 
weeks apart b capsular antigen in separate and 
OMP antigen in separate rabbit groups. This 
based on the theme that immuoadjuvant can be 
of use either pre,mix with or post-antigen priming 
to an immune animal model (Zhao et al., 2023). 
Both of the prepared antigens bear forigeness 

characters from the organism producing them 
that are encoded by the genetic system of that 
organism (Venter, 1998). Parallel to this the 
immune system cells of test animal model are 
encoded by gene sets that on their expression, 
these cells recognize that the introduced material 
facing them are foreign (Lagou et al., 2018). 
Immunogenicity in theoritical immunology sense 
is a matter of self/non-self recogntion theme 
(Pradeu & Carssella 2006). Both of capsule b 
and OMP were found to be as lapin immunogens 
inducing humoral immune responses both at 
systemic [blood sera] and mucosal [appendex 
globulin] levels. The immune conversion were 
estimated by the folds increase of antibody titre 
and concentrations than the baseline titre and 
concentrations from the baseline immune 
function in normal control rabbits. Both of 
systemic anticapsular and anti-OMP were 
express five to six folds increase. While for 
mucosal antibody titres rise was four to five folds 
increase than normal immune function was five 
folds. Capsular b antigen induce leukocyte 
stimulatory factor up to 1.08% as compared to 
saline control was 0.95%, which is a kind of 
cytokine that promote leukocyte migration in 
capillary tube approach (Sixt & Lammermann 
2011; Thorley et al. 2007). 
 
Both of the Capsule b and OMP antigens does 
not express delayed allergenic immune 
conversions than the baseline control rabbits. 

 
Table 1. Cellular Immune functions as leukocyte Inhibitory factor cytokines[A] and Skin 

DTH[B] for H. infleunzae b 
 

A Specific Immune 
Primed Rabbit 

Systemic Leukocyte 
Inhibitory Factor 

Mucosal Leukocyte 
Inhibitory Factor 

Capsule primed R1 
R2 
R3 
Rmean  
Control 

1 
1 
1 
1 
0.98 

1 
1.09 
1.07 
1.08 
0.995 

OMP primed R1 
R2 
R3 
Rmean 
Control 

1 
0.9 
0.9 
0.903 
0.98 

1 
0.95 
0.91 
0.95 
0.97 

B/Skin DTH/hours Erythema Induration Necrosis 

Capsule primed 
6  
48 
72 
OMP primed 
6 
48 
72 

 
- 
- 
- 
 
- 
- 
- 

 
- 
- 
- 
 
- 
- 
- 

 
- 
- 
- 
 
- 
- 
- 



 
 
 
 

Shnawa et al.; Asian J. Immunol., vol. 8, no. 1, pp. 1-7, 2025; Article no.AJI.129951 
 
 

 
5 
 

Table 2. Humoral Immune Function for capsular [A] and OMP [B] primed rabbits as precipitins 
A and hemagglutinins B. For the neurogenic H. infleunzae b 

 

Animal 
groups 

Capsule 
Primed 
rabbits 

Serum 
antibody titre 
means 

Serum 
concentration 
means gm/L 

Mucus titre 
means 

Mucus 
concentration 
means gm/L 

Test Group 
A 

R1 
R2 
R3 
Rmean 
Control 

320 
640 
640 
533.3 
10 

26.3 
31.42 
32.1 
29.94 
6.75 

64 
32 
32 
42.6 
2 

1 
2.7 
2.3 
2.06 
0.45 

Test group  
B 

OMP Primed 
R1 
R2 
R3 
Rmean 
Control 

 
1280 
640 
640 
853.3 
10 

 
33.1 
21.2 
22.31 
25.4 
6.35 

 
64 
128 
64 
85.33 
2 

 
2.7 
1.0 
1.0 
1.56 
0.4 

 
Since they neither produce typical skin DTH 
reaction nor Jone-Moote reactions (Burrell, 
1979), during the observation period 6 up to 72 
hrs. Post to sensitin intradermal injection in 
primed test animals groups. 
 
The possible epitope nature of capsular b 
antigen may be T independent epitope triggering 
anti-PRP antibodies. While that of OMP 
antibodies may trigger TH2 cells and Th2 cells in 
turn activate B cells to produce anti-OMP 
antibodies. Apparently, it does not contain TH1 
dependent epitopes but there is a possibility for 
shiffting from TH2 to TH1 dependence due to co-
existing epitopes mixed with independent ones 
(Misseneir & Lary 2002; McVernon et al., 2005; 
Shnawa, 2006; Haddadi et al., 2022; Garcia-Diaz 
et al., 2013; Jackson et al., 2024). Thus type b 
capsule and OMP of H. infleunzae induced local 
and systemic humoral immune responses in 
rabbits primed with them separately. Systemic 
humoral antibody responses were higher than 
that mucosal responses. This was inline with 
Shnawa (2006), working on immunogenicity of C. 
fetus in rabbits. The ratio of systemic to mucosal 
were 10;1 in term of titre while in term of 
concentration for OMP was 13:1 and for capsule 
was 14:1 (Shnawa, 2006). 
 

5. CONCLUSIONS 
  
Human neurotropic neurogenic local H. 
infleunzae b OMP and Capsule b are found as 
lapin immunogen. Adult rabbits proved to be 
valid immune model for testing immunogenicity 
of these antigens. Capsular b antigen induce 
leukocyte stimulatory factor in capillary method. 
Capsular and OMP induces humoral 
hemagglutinin and precipitin responses both at 

systemic and mucosal compartments. Such 
findings be essential for preclinical and 
development of systemic and/or mucosal 
vaccine, on approval will be valid for local mass 
vaccination of childhood meningitis. 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 

Authors herebydeclare that NO generative AI 
technologies such as Large Language 
Models(ChatGTP,COPILOT, etc ) and text-to-
image generators have been used during wrting  
or editing of this mnanuscript.  
 

CONSENT AND ETHICAL APPROVAL 
 

It is not applicable. 
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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© Copyright (2025): Author(s). The licensee is the journal publisher. This is an Open Access article distributed under the terms 
of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, 
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Peer-review history: 
The peer review history for this paper can be accessed here: 

https://pr.sdiarticle5.com/review-history/129951  

https://pr.sdiarticle5.com/review-history/129951

