







































_____________________________________________________________________________________________________ 
 
*Corresponding author: E-mail: ibrahimshnawa3@gmail.com; 
 
Cite as: I M S, Shnawa, and AL-Janabi AAJ. 2024. “Immune Characterisation of Human Local Pneumotropic Isolates of 
Streptococcus Pneumoniae Serotype 1 and 6 As Local Vaccine Strains”. Asian Journal of Immunology 7 (1):285-91. 
https://doi.org/10.9734/aji/2024/v7i1151. 
 

 
 

Asian Journal of Immunology 
 
Volume 7, Issue 1, Page 285-291, 2024; Article no.AJI.127471 
 

 
 

 

 

Immune Characterisation of Human 
Local Pneumotropic Isolates of 

Streptococcus pneumoniae Serotype 1 
and 6 as Local Vaccine Strains 

 
Shnawa I M S a,b* and AL-Janabi  AAJ c 

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

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

c Department of Biology, College of Science, University of Kufa, Najaf, Iraq. 
 

Authors’ contributions  
 

This work was carried out in collaboration between both authors. Both authors read and approved the 
final manuscript. 

 
Article Information 

 
DOI: https://doi.org/10.9734/aji/2024/v7i1151 

 
Open Peer Review History: 

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Received: 10/10/2024 
Accepted: 10/12/2024 
Published: 17/12/2024 

 
 

ABSTRACT 
 

S. pneumoniae is one of the principle human pneumopathogen. Such pathogenicity features in man 
can be delineated by severity, capsule nature and yield of pure growth onto primary plate cultures. 
While in laboratory animal models, it accounts for; the nature of capsule serotype, shortness of time 
for the death end point and extent of tissue pathology. The virulent serotypes   C1 and C 6 of 
human pneumotropic S. pneumoniae were immune characterized as vaccine strains. Capsules 
were separated, identified and quantified. Then dispensed as 10mg deried amounts in ampoules. 
On reaching specific immune priming of rabbits the ampoules dissolved in 10 mls sterile saline and 

Original Research Article 

https://doi.org/10.9734/aji/2024/v7i1151
https://www.sdiarticle5.com/review-history/127471


 
 
 
 

Shnawa and AL-Janabi; Asian J. Immunol., vol. 7, no. 1, pp. 285-291, 2024; Article no.AJI.127471 
 
 

 
286 

 

each ml admixed with 1ml lanoline and injected intramuscularly per each rabbit's thigh muscles. The 
immune primed rabbits left for 28 days then test bleed to check for an evident immune conversion 
then, at the day 35 after immunization the immune rabbits were challenged with live S. pneumoniae 
1x 10 to six CFU/ml., in a rate of 2 mls. The carbohydrate based capsule prototype monvalent 
vaccines were found as; Pure, safe, antigenic, immunogenic and immune protective to the rat of 
80% for C1 and 60% for C6 serotype in rabbit models. They induced systemic humoral immune 
precipitins responses higher than mucosal pricipitin responses. Proinflammatory cytokine responses 
TNF alpha, TNF beta and IL6 and anti-inflammatory cytokine IL10 were mounted as mucosal 
responses higher than the systemic responses. The proved immune characteristics of these 
capsule serotypes were suggestive for use of these strain as vaccine strains along with other 
serotypes in development of the local prototype multiserotype vaccines of this pathogen. 
 

 
Keywords: Antigenicity; cytokine; immunogenicity; immune efficacy; proinflammatory precipitins; 

vaccine strain. 
 

1. INTRODUCTION 
 

The nature of capsule serotype in S. pneumoniae 
played as a determinal factor in pathogenesis, 
pathogenicity and immunogenicity [1-10]. S. 
pnenumoniae in our previous work in this area, 
have shown inter and intraserotype variations in 
pathogenicity to mice and rabbits [10]. So far the 
immune characterisation of the sertype 1 and 6 is 
concerned as a vaccine strains [10]. There were 
few bacterial specific carbohydrate based 
vaccine that are approved for mass vaccination 
in human clinical practice of bacterial infectious 
diseases. These vaccines are avialable in use for 
those at risk human beings by the vaccine 
approval authorities [1]. Such as that of multiple 
carbohydrate based vaccine of S. pneumoniae 
[2-10]. Though at times human pneumococcal 
pneumonia reported every now and then at 
diferent part of the developing world countries 
[10]. So local human pneumotropic S. 
pneumoniae isolates gained prevelence may 
express different pathogenic and immunogenic 
potentials. Such differences may be from the 
same serotypes present in the current 
internationally approved vaccine [10,11]. The 
present paper was aims at tempts to immune 
characterize human pneumotropic local serotype 
1 and 6 S.pneumoniae isolates as local vaccine 
strains. 
  

2. MATERIALS AND METHODS 
 

Seed Strains: A collection of eight serotypes of 
S. pneumoniae that were recovered from clinical 
human cases of pneumonia. The study strains 
were elected following certain criteria both in 
man and laboratory animals. In man, serotype 
nature, severity while in animal model, serotype 
nature, shortness in death end point time and 
extent of tissue pathology. The most virulent of 
which were serotype 1 and 6 so they were eleted 

for immune characterisation as vaccine strains 
[10]. 
 
Laboratory Development: The 24 hr freshly 
revived original isolates of each serotype 1 and 6 
[author collection [10] of the S. pneumoniae 
serotype 1 and 6 growth were made onto blood 
agar plates. Sterile heavy swab inocula were 
made onto 20 trypicase soy agar plates for each 
serotype. The inoculated plates were incubated 
at 5-10% CO2 tension at 37C for 24 hrs. Pure 
growth were harvested by 5 ml sterile saline 
solution per each plate. The suspensions were 
brought to PH 8.5 by the addition of 10% KOH. 
The alkalinized suspensions were heated at 90C 
for 1hr in water bath. These heated suspensions 
let stand to cool and acified with 5N acetic acid. 
The acidified suspensions then centrifuged at 
5000 rpm for 10min.Pellets were discarded and 
supernatant s were mixed with two volumes of 
1% sodium acetate in 95% ethanol solution then 
kept at 4C for 24 hrs. Supernate and pellets were 
formed. The formed precipitates were removed 
by centrifugation at 2500rpm for 10 min. Pellets 
were discarded and supernatants were mixed 
with 5% sodium actate in 0.5 N acetic acid for 24 
hrs at 4C. These mixed solutions were 
centrifuged at 5000 for 30 min. Supernatant 
discarded and pellets were washed with 
85%,95% absolute ethanol. Then dried at 37C. 
The 20 plate yeilds 200 mg powder materials. 
These materials were cautiousely dispensed in 
10 mg per ampoule under aceptic precautions 
and kept till use. Each ampoule dissolved in 10 
mls., sterile saline representing the immungenic 
doses for 10 rabbits and stands as prototype 
monovalent capsular carbohydrate based 
experimental vaccine in rabbits [12]. 
 
Purity: A purity checks were done by quadrate 
streak method onto trypticase soy agar plates in 



 
 
 
 

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287 

 

each step of the laboratory developmennt of the 
prototype monovalent pneumococcal vaccine 
[13]. 
 
Safety: A 0.1 ml of the capsule experimental 
prototype vaccines were IM injected into rabbits 
through thigh muscles three replicates for each 
serotype. The injected animals were followed for 
up to five days. Then eviscerated to test for gross 
and histology changes [13]. 
 
Adjuvant: Lanolin solution [13]. 
 
Immunogens: One volume of lanolin was 
admixed with one volume of 1mg/ml. capsular 
polysaccharide. The mixture stands as the test 
immunogens [13]. 
 
Immunization Protocol: The immunogen 2 ml 
mixture was IM injected into the thigh mscle of 
rabbit and left for 15 days then test bleed [13]. 
 
Laboratory Animals: A group of Newzeland 
rabbits were acclimatzed for housing conditions 
for two weeks and kept ad libitum cnodition 
during specific immune priming and challenge 
experiments. These rabbits were categorized into 
the following groups; 
 
Safety group........................................6 Rabbits 
Serotype 1 specific immune priming...10Rabbits 
Serotype 6 specific immune priming...10 Rabbits 
Sham Control .......................................10 rabbits 
 
Challenge Models: The specific immune primed 
rabbit groups at the day 22 postpriming were 
challenged with live S. pneumoniae in strenght of 
1x10 to six /m, survivivors were scored 21 days 
post challenge [14] 
 
Blood Sampling: Test and control rabbits 
groups were subjected to blood collection by 
cardiac puncture. Sera were saved, dispensed at 
0.5 ml alqouts in appendroph tubes and kept at -
20 till test for antibody and cytoikine levels [15] 
 
Tracheal Mucosal Globulins: Parts of the 
challenged and control rabbits trachea were 
incised and open up into sterile petri –plates. 
Tracheal mucasa were scrapped into the plates 
then 5 ml formal normal 0.5% saline were added 
to the scraps and mixed thorughly and tubbed 
into centrifuge tubes. Scraps saline tubes were 
centrifuge at 5000 rpm for 10 min. Supernatants 
were kept for processing of mucosal globulin 
separations in accordance with the method [16]. 
Pellets were discarded 

Immune Investigations: Determinations of 
antibody response levels were done by 
precipitation tests as in [17,18]. Cytokine 
response measurements were done as the 
manufaturer instrutions. 
 
Efficacy Percent Difference Statistics: Let the 
efficacy percent of Serotype 1 Is a and Serotype 
6 is b .So the Efficacy difference percetage is: 
 

[a-b]/[a+b/2] x 100 
 

3. RESULTS 
 
Purity: All of the laborsatory development purity 
checks onto trypticase soy agar plates were 
found of negative growths. 
 
Safety: Neither gross nor histologic changes 
were noted in the inoculated safety check 
rabbits. 
 
Antigenicity: One from each of the vaccine lot 
ampoules were used for check of antigenicity test 
by precipitation tests. 
 

3.1 Immunogenicity 
 
Humoral Antibody Responses: The mean of 
serum and mucosal antibody titres for the 
challenged rabbits were 400 and 20 for serotype 
1 and 213.33 and 13.33 for serotype 6. Systemic 
responses were higher than mucosal responses, 
Table 1. 
 
Cytokine Responses: Mucosal cytokine 
responses were higher than systemic cytokine 
responses both for the serotypes 1 and 6.,        
Table 2. 
 
Cytokine imbalance: Cytokine imbalance were 
noted in serotype 1 vaccine at mucosal response 
only, Table 3. 
 
Immune Efficacy: The immune efficacy of 
sertype 1 was 80% and for serotype 6 was 60%, 
Table 4. The efficacy percenatge difference is 
equal to 28.571%.Since practically good vaccine 
is that gaving around 90% protection and the 
efficacy percetage difference approaching one 
third .Thus serotype C 1 is more immune 
protective than serotype 6 in local multivalent 
candidate pneumococcal vaccine taking in 
consideration the limits of immune interference. 
 
Prototype Carbohydrate Based Vaccine 
Features: S. pneumoniae serotype 1 prototype 



 
 
 
 

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288 

 

vaccine was found; Pure, safe, antigenic,                     
weak immunogenic needs exogenous adjuvant, 
induce humoral systemic precipitin responses 
higher than mucosal responses. Mucosal 
Cytokine responses were higher than systemic 
responses. Induce cytokine imbalance at 
mucosal surfaces but not at systemic 
compartment. It was immune efficus to 80% in a 
lapin challenge model. Serotype 6 prototype 

vaccine was found; Pure, safe, antigenic, week 
immungenic need exogenous adjuvant. Induce 
systemic humoral immune resoponses higher 
than mucosal responses. Mucosal cytokine 
responses were higher than the systemic 
responses. It neither induce cytokine imbalance 
at mucosa nor at systemic compartment. It was 
immune efficus to 60% in lapin challenge model, 
Table 5. 

 
Table 1. Specific immune precipitins respnses of the immne challenged rabbits 

 

Groups Titres 

Serotype 1 specific immune primed rabbits 
Mucosal response 
Systemic response 

 
20 
400 

Serotype 6 specific immune primed rabbits 
Mucosal response 
Systemic response 

 
13.33 
213.33 

Control rabbits 
Mucosal response 
Systemic response 

 
3.33 
6.3 

 
Table 2. Cytokine responses of immune primed challenged rabbits 

 

Rabbit groups TNF alpha TNF beta IL6 IL10 

Serotype 1 specific 
immune primed 
challnged rabbits 
Mucosa 
Systemic  

 
 
 
473.11-+29 
250-+30.7 

 
 
 
387.21 -+1.39 
250.11-+7.6 

 
 
 
360.59-+4.3 
223.5-+20.2 

 
 
 
253.36-+23.5 
202.6 -+14.5 

Serotype 6 specific 
immune primed 
challenged rabbits 
Mucosa 
Systemic 

 
 
 
233.55-+1.4 
245.2-+35 

 
 
 
225.25-+7.3 
236.32-+27.8 

 
 
 
238.8-+4.9 
219.51-+59.6 

 
 
 
199.68-+13.5 
200.76-+8.1 

Control 
Mucosa 
Systemic 

 
125.12-+55 
115.29-+11.7 

 
110.35 -+11.5 
101 -+11.5 

 
93.54 -+2.5 
100.62-+0.75 

 
110.25 -+ 11.5 
101.15 -+11.5 

 
Table 3. S. pneumoniae capsular 1 and 6 primed challenged rabbits cytokine imbalance 

 

Rabbit groups TNF alpha TNF B IL6 IL10 

Sertype 1 specific immune primed challenged 
rabbits 
Mucosa 
Systemic 

 
 
4:1* 
2:1 

 
 
3:1 
2:1 

 
 
3:1 
2:1 

 
 
2:1** 
2:1 

Serotype 6 specific immune primed challenged 
rabbits 
Mucosa systemic 

 
1.5:1 
1:1 

 
2:1 
1:1 

 
2:1 
2:1 

 
2:1 
1:1 

Control 
Mucosa/ 
systemic 

 
 
1:1 

 
 
1:1 

 
 
1:1 

 
 
1:1 

• The number of folds of cytokine concentrations for the test to control rabbits 
** Imbalance is the number of concentration folds for the proinflammatory to the anti-inflammatory cytokines. 

 
 



 
 
 
 

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289 

 

Table 4. The immune efficacy of the developed vaccines in rabbits 
 

Prototype 
vaccine 

Infective dose Number of 
rabbits 

Servive to total Percentages 

Type 1 1x10 to 6 10 8:10 80% 
Type 6 1x10 to 6 10 6:10 60% 
Control 1x10 to 6 10 0:10 0% 

 
Table 5. Features of the Carbohydrate based S. pneumoniae serotype 1 and 6 monotypic 

prototype vaccines in rabbit challenge model 
 

Features Protype C1 Prototype C6 Approved 
pneumococcal Vaccine 

Understanding 
Disease 
Understaning causal 

 
Pneumonia 
S. pneumoniae 

 
Pneumonia 
S. pneumoniae 

 
Pneumonia 
S. pneumoniae 

Virulence factor Serotype 1 Serotype 6 23 serotypes 

Purity Pure Pure Pure 

Safety Safe Safe Safe 

Antigenicity Antigenic Antigenic Antigenic 

Immunogenicity  Immungen*  Immunogen*  Immunogen* 

Immune efficacy in 
rabbit 
Immune efficacy in 
man 

 
80% 
 
? 

 
60% 
 
? 

 
60% 

• Amplified on combination with lanlin 

 

4. DISSCUSSION 
 

The subunit bacterins of the bacterial pathogens 
when introduced to the body of a small mammal 
laboratory animals like rabbits, Tables 1 – 5, may 
triggers TH1, TH2 and /or B cells to grow, 
proliferate, expand and activated as an effector 
and/ or memory cells. As a consiquences, 
humoral mucosal and systemic antibody 
responses as well as cytokine network 
activations both for innate and adaptive cytokine 
types [19,20] The systemic humoral immune 
responses were higher than mucosal responses 
[16]. While the mucosal cytokine responses were 
higher than the systemic responses [4]. Cytokine 
imbalance was noted betwen pro and anti-
inflammatory cytokines serotype 1 at mucosal 
compartment but not systemic. Serotype 6 does 
not express imbalance at both mucosal and 
systemic compartments [21]. The immune 
effcacy of the present prototype carbohydrate 
based capsular bacterins were ranging between 
60- 80% [11,22]. The vaccinologic features of the 
prepared candidates capsular bacterins were; 
Antigenic, weak immunogenic need an 
exogenous adjuvant leading to antibody and 
cytokine responses. Such responses were both 
at mucosal and systemic compartments with an 
immune efficacy ranging betwen 60 to 80% in 
rabbit models. The proposed S. pneumoniae 

vaccine strains may be of help in development of 
an eight local pneumococcal vaccine with the 
other reported six serotypes [10] which may be 
more efficieous in this area than the 
internationally approved 23 pneumococcal 
vaccine [22]. The vaccine strains form an integral 
part in vaccine development, since it forms the 
first step in vaccine development processes. S. 
pneumoniae serotype 1 and 6 are                         
among the known serotypes ensembled in 
various up date and approve vaccine 
formulations [23,24]. 
 

5. CONCLUSION 
 

Local pneumotropic isolates of S. pneumoniae 
serotype 1 and 6 were found valid as vaccine 
strains based on lapin immune challenged 
models. The proved immune characteristics of 
these capsule serotypes were suggestive for use 
of these strain as vaccine strains along with other 
serotypes in development of the local prototype 
multiserotype vaccines of this pathogen. 
 

DISCLAIMER (ARTIFICIAL INTELLIGENCE) 
 

Authors hereby declare that NO generative AI 
technology Models (Chat GPT, COILOT, etc) and 
text-to-image generator have been used during 
writing or editing of this manuscript. 



 
 
 
 

Shnawa and AL-Janabi; Asian J. Immunol., vol. 7, no. 1, pp. 285-291, 2024; Article no.AJI.127471 
 
 

 
290 

 

CONSENT 
 
It’s not applicable. 

 
ETHICAL APPROVAL 
 
Care, housing, handling and managment done 
on rabbits were following the international acts 
regulating care, housing, handling and 
intervensions. 

 
COMPETING INTERESTS 
 
Authors have declared that no competing 
interests exist. 

 
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