Human vaccine candidates for infections caused by Klebsiella pneumoniae: A systematic review

Abstract Background and Aims There are many difficulties in treating Klebsiella pneumoniae, necessitating the creation of more preventative/therapeutic measures like vaccinations. However, after numerous attempts, there is still no authorized and widely accessible vaccine. The present study aimed to systematically review published studies on K. pneumoniae vaccines in human subjects/samples. Methods To find published studies, several electronic databases, including Scopus, PubMed, Web of Science, ClinicalKey, ClinicalTrials.gov, and Cochrane Library were searched without time limitation using the appropriate keywords. Studies were scrutinized, and the information from those that met our inclusion criteria was gathered and analyzed. Results In total, 691 studies were found, of which 14 articles were included for systematic review. Bacterial lysate containing K. pneumoniae was the most studied vaccine candidate. As the main indicator of human immune responses to K. pneumoniae, antibody responses were determined by most studies. The antigen amount, the route of immunization, and the immunization schedule were varying in the studies and were chosen based on several factors such as the disease model, the vaccine type, the vaccination setting (prophylactic or therapeutic), and so on. Conclusion The majority of studies asserted that their vaccination was efficient and safe, which was demonstrated by a decrease in the rate of infections and the induction of protective antibody, cell‐dependent, and/or cytokine responses. Altogether, the information provided here will help researchers examine the K. pneumoniae vaccine candidates more closely and take future actions that will be more consistently successful.


| Inclusion and exclusion criteria
The following were the inclusion criteria: (1) Research focused on developing vaccines (either therapeutic or prophylactic vaccines) to protect people from K. pneumoniae infections, (2) Clinical and preclinical trials, and experiments using human subjects or human samples, (3) Studies that used a K. pneumoniae-derived vaccine, or a part of its vaccine, include K. pneumoniae, (4) Published studies in English.
The following were the exclusion criteria: (1) Animal studies and studies without human subjects or samples, (2) Narrative/systematic reviews, letters, books, conferences, and comments, (3) Non-English, duplicate, and non-full text available articles, (4) Research without experimental results (e.g., in silico studies), (5) Studies using autovaccines that designed a vaccine based on the patients' bacterial culture, hence their vaccine component varied accordingly.

| Data collection
Several data were gathered from the selected studies as follows: authors, immunization setting, published year, country, infection's type, vaccine type, vaccine name, vaccine components, adjuvant, human population, sample type, sample size, control, gender, age, control, the amount of antigen used, route, schedule, immune response assay, safety, and the main outcomes.Two authors separately evaluated the articles' quality, and MR then reviewed the results to resolve any inconsistencies.

| RESULTS
Following the search strategy, a total of 691 records was found, of which 140 were duplicates and hence were excluded.Of 551 remained studies, 452 records were excluded with reasons by title and abstract reading.Based on our exclusion criteria, 55 articles were excluded and 44 studies remained for eligibility assessment.After the eligibility step, 30 more studies were excluded and finally, 14 articles remained for systematic review (Figure 1).

| General data
Of 14 included studies, seven were published before 2000, and seven were published after 2000.Four studies were performed in the USA, three studies were done in France and Spain, and one study was conducted in each Bulgaria, Portugal, Switzerland, and UK.Individuals receive therapeutic vaccinations after they have already been exposed to the infection, whereas prophylactic vaccines are given to people as a preventative strategy to avoid the infection.
In the present study, the setting was equal for prophylactic and therapeutic vaccination (seven studies for each).
The most sample used was sera samples (six studies), followed by saliva samples (two studies), and one study of each of peripheral blood mononuclear cells (PBMCs), bronchus biopsy, cervical-vaginal secretions, throat sample, tonsil sample, and urine.

| Vaccines type/components
The most used vaccine type was bacterial lysate containing K.  (Continues) The populations used differ based on the vaccination setting (prophylactic or therapeutic vaccination).In six out of seven prophylactic studies, healthy volunteers were vaccinated, while in one prophylactic study, victims of acute trauma were used.The mean sample size was 39.83, and mainly adults were used.In four prophylactic studies both genders were used, while in two studies only men were used.
In therapeutic studies, four studies were done on UTI patients, and one study was done for each of children who required tonsillectomy, patients who had undergone pneumectomy for cancer, and recurrent RTI patients.The mean sample size was 369.57and the vast range of ages was used.The most therapeutic studies were done on women with recurrent UTI (four studies), while in one study both genders was used and the gender was unknown in two studies (Table 1).

| Immunization
The amount of antigen for immunization was only reported in seven studies and was varying based on the vaccine type.
Except one study, the booster dose/doses were used in the studies.The immunization schedule was varying based on the vaccination setting and the vaccines type (Table 1).
Antibody response had been assayed in the majority of studies (nine studies) as the main indicator of K. pneumoniae-specific immune response.Other arms of immune response were also studied in some studies as follows: T cells activation (three records), cytokines (three records), and innate immune responses (one record) (Table 1).

| Efficacy/effectiveness of used vaccines
The effects of K. pneumoniae vaccine candidates on human subjects/samples were determined by two factors, first, the immune response, and second the protection.The protection (i.e., the reduction in the infection rate and/or severity) was reported in seven studies in which five studies reported a relatively protective effect of their vaccine, while two studies did not observe protection in their vaccinated groups.This non-protective effect was seen for K1 CPS (a polysaccharide vaccine) and a bacterial lysate of K.  1).

| Safety
The safety of used vaccines was only reported in five studies.No adverse effect was seen in any of these five studies, hence the vaccines were all safe (Table 1).

| DISCUSSION
In the current study, we conducted a systematic review of all studies on human vaccines against K. pneumoniae infections.The number of studies that were included was 14 studies, which is a small quantity, particularly given the fact that there were no time limits.The relatively small number of studies may be attributed to the difficulty and expensive approaches to K. pneumoniae vaccination, the inadequate K. pneumoniae antigens as vaccine candidates, the safety issue, and several more challenges of K. pneumoniae vaccination.Two of several challenges in developing K. pneumoniae vaccines are the most significant ones; first, there should be no cross-reactivity between the vaccine and the natural flora of the gut, where K. pneumoniae colonizes, 27 Second, the majority of K. pneumoniae-vulnerable individuals are older and have compromised immune systems as a result of various comorbidities, which has resulted in decreased or nonexistent vaccine protective responses. 28eviously, a few vaccines were developed and tested in humans. 8erefore, it was expected that much more studies be found on humans, but surprisingly only 14 relevant studies were found in the databases.In addition to the limitations mentioned above about difficulties of K.
pneumoniae vaccination, the low number of human studies may refer to the ineffectiveness of the previously-introduced vaccines in afterward evaluations.It should be also noted that the sample size of many of these 14 human studies was low which undermine their obtained results.In addition, half of the human studies on K. pneumoniae vaccination were done before 2000, and only three studies were done in the last 10 years.
This also shows the reluctance of researchers to conduct K. pneumoniae vaccination studies in humans.Considering the importance of K. pneumoniae vaccination (at least for people at risk), more favorable conditions should be provided to conduct much more studies in this field.It is noteworthy that some clinical trials are ongoing in this field that were not included in our study since their data is not fully available yet.
Out of 14 studies included in our systematic review, 10 studies used bacterial lysates, and eight of them showed degrees of protection against K. pneumoniae.Although whole cells vaccines are usually considered as main inducers of immune responses, their most significant challenge is the issue of safety, demanding new antigens and/or strategies to develop safer vaccines. 29,30Other studies in our systematic review used ribosomes, polysaccharides, and glycoprotein.
The immune system's most powerful weapons against K. pneumoniae are antibodies, which may attach to its surface molecules, neutralize it, and facilitate phagocytosis. 31This mechanism is known as opsonization, by which phagocytes recognize the Fc region of the antibodies coating the pathogen. 32Because of this, most of the studies employed antibody responses as the primary measure of immunological responses.T cell-dependent responses that are mediated by cytokines, in addition to antibody responses, are crucial for producing complete protection against K. pneumoniae, although their precise functions are still unclear. 10e antigen amount, the route of immunization, and the immunization schedule were varying in the studies.However, it was expected, because these characteristics depend on a number of variables, including the disease model, the vaccine type, the vaccination setting (prophylactic vs. therapeutic), and so on.As previously reported, 33 infection dose is a determining factor that may alter the intensity or direction of immune responses, and as a result, alter the infection's final outcome.Therefore, if it is possible, it is recommended that a pilot study be performed before immunization to find the best dose and route.
The majority of our analyzed studies claimed a protective and safe K.
pneumoniae vaccine candidate.However, since studies indicating inefficient, non-protective, or hazardous vaccines tend to be more rejected by journals than studies with positive findings, publication bias may have occurred in this field, especially since most of the studies were published a long time ago, but no significant progress has been reported afterward, and none of them have been approved for use until now.The good news is that several of these studies present a potent and safe K.
pneumoniae vaccine that is suggested to be further studied to potentially defeat this life-threatening pathogen.
As a limitation of our study, it should be mentioned that we excluded books, letters, conferences, and comments.The most accurate K. pneumoniae vaccination studies were found with the use of this exclusion, but it may have left out some less well-known but yet potent vaccine candidates.

| CONCLUSION
Altogether, in the present study, we thoroughly examined all available information on vaccine candidates of K. pneumoniae tested in human.This information includes information on antigens, immunization setting and schedule, safety, and the results of earlier research.The gathered and well-clustered data of the present systematic review would help researchers to evaluate the potential K. pneumoniae vaccines more thoroughly and to make a more fruitful future.
pneumoniae with 10 records.Other vaccine types include bacterial ribosomes (two records), polysaccharides (two records), and glycoprotein (one record).The bacterial lysate studies have used inactivated or killed lysate of several bacteria including K. pneumoniae.Accompanied with K. pneumoniae, these bacteria are all involved in the K. pneumoniae-caused infections.Both records of bacterial ribosomes used ribosomal fractions of K. pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, and Streptococcus pyogenes.The study of glycoprotein and both studies of polysaccharides used capsular polysaccharide (CPS) as the main component of their vaccines.Adjuvant was used only in one study which applied K. pneumoniae proteoglycan as adjuvant beside bacterial ribosomes vaccine (Table 1 ). Flowchart of the study selection.The characteristics of human studies on Klebsiella pneumoniae vaccine candidates.