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Updated: May 5, 2026
Assessing Respiratory Immune Responses to Haemophilus Influenzae
Published on: June 29, 2021
A practical method for preparation of pneumococcal and nontypeable Haemophilus influenzae inocula that preserves
Lea-Ann S Kirkham1, Karli J Corscadden, Selma P Wiertsema
1School of Paediatrics and Child Health, The University of Western Australia, 35 Stirling Highway, Perth, WA 6009, Australia. lea-ann.kirkham@uwa.edu.au.
Background:
Convenience is a major reason for using killed preparations of bacteria to investigate host-pathogen interactions, however, host responses to such preparations can result in different outcomes when compared to live bacterial stimulation. We investigated whether cryopreservation of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) permitted investigation of host responses to infection without the complications of working with freshly prepared live bacteria on the day of experimental challenge.
Findings:
S. pneumoniae and NTHi retained >90% viability following cryopreservation in fetal calf serum for at least 8 weeks. Host responses to live, cryopreserved (1 week and 4 weeks), heat-killed or ethanol-killed S. pneumoniae and NTHi were assessed by measuring cytokine release from stimulated peripheral blood mononuclear cells (PBMCs). We found that cryopreserved bacteria, in contrast to heat-killed and ethanol-killed preparations, resulted in comparable levels of inflammatory cytokine release from PBMCs when compared with fresh live bacterial cultures.
Conclusion:
Cryopreservation of S. pneumoniae and NTHi does not alter the immunostimulatory properties of these species thereby enabling reproducible and biologically relevant analysis of host responses to infection. This method also facilitates the analysis of multiple strains on the same day and allows predetermination of culture purity and challenge dose.
Insights
Cryopreservation of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) maintains bacterial viability and preserves immunostimulatory properties. This method allows for reproducible host response analysis without complications from live bacteria.
Area of Science:
- Microbiology
- Immunology
- Infectious Diseases
Background:
- Investigating host-pathogen interactions often uses killed bacteria for convenience.
- However, killed bacteria may yield different host responses compared to live bacteria.
- Cryopreservation offers a potential alternative for preserving live bacteria for infection studies.
Purpose of the Study:
- To determine if cryopreservation of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) allows for investigation of host responses.
- To assess if cryopreserved bacteria elicit comparable host responses to live bacteria.
Main Methods:
- Streptococcus pneumoniae and NTHi were cryopreserved in fetal calf serum.
- Bacterial viability was assessed after cryopreservation.
- Peripheral blood mononuclear cells (PBMCs) were stimulated with live, cryopreserved (1 and 4 weeks), heat-killed, or ethanol-killed S. pneumoniae and NTHi.
- Cytokine release from PBMCs was measured to assess host response.
Main Results:
- Cryopreservation maintained >90% viability for S. pneumoniae and NTHi for at least 8 weeks.
- Cryopreserved bacteria elicited inflammatory cytokine release from PBMCs comparable to fresh live bacteria.
- Heat-killed and ethanol-killed preparations resulted in different cytokine release profiles compared to live bacteria.
Conclusions:
- Cryopreservation of S. pneumoniae and NTHi does not alter their immunostimulatory properties.
- This method enables reproducible and biologically relevant analysis of host responses to infection.
- Cryopreservation facilitates standardized experimental conditions, including strain analysis and challenge dose determination.
