Infect Dis Ther (2021) 10:2157–2175

https://doi.org/10.1007/s40121-021-00533-4

REVIEW

Current Stage in the Development of Klebsiella

pneumoniae Vaccines
Lucas Assoni . Raquel Girardello . Thiago Rojas Converso .
Michelle Darrieux

Received: May 17, 2021 / Accepted: August 24, 2021 / Published online: September 2, 2021
Ó The Author(s) 2021

ABSTRACT formulations are discussed. The contribution of

antibody and cell-mediated responses is also
Klebsiella pneumoniae is a bacterium capable of presented. In summary, K. pneumoniae vaccines
colonizing mucous membranes, causing serious are feasible and a promising strategy to prevent
infections. Widespread antibiotic resistance in infections and to reduce the antimicrobial
K. pneumoniae—either through intrinsic mech- resistance burden worldwide.
anisms or via acquisition from different species,
especially in hospital environments—limits the
therapeutic options against this pathogen, fur- Keywords: Klebsiella pneumoniae; Vaccines;
ther aggravating the disease burden. To date, Vaccination coverage; Enterobacteriaceae
there are no vaccines available against K. pneu-
moniae infection. Although formulations based Key Summary Points
on capsular polysaccharides have been pro-
posed, the high variability in capsular serotypes Klebsiella pneumoniae infections represent
limits vaccine coverage. Recombinant vaccines a major public health threat.
based on surface exposed bacterial antigens are
a promising alternative owing to their conser- Different vaccine strategies against
vation among different serotypes and accessi- K. pneumoniae have been investigated and
bility to the immune system. Many vaccine some have reached clinical trials.
candidates have been proposed, some of which
The complexity of K. pneumoniae
have reached clinical trials. The present review
infections suggests multicomponent
summarizes the current status of K. pneumoniae
vaccines as a promising approach to
vaccine development. Different strategies
provide long-term immunity with wide
including whole cell vaccines, outer membrane
coverage.
vesicles (OMVs), ribosome, polysaccharide,
lipopolysaccharide (LPS), and protein-based New technologies are an interesting
strategy to refine selection of novel
vaccine candidates.
L. Assoni
R. Girardello
T. R. Converso

M. Darrieux (&)
Laboratório de Biologia Molecular de
Microrganismos, Universidade São Francisco,
Bragança Paulista, Brazil
e-mail: [email protected]
2158 Infect Dis Ther (2021) 10:2157–2175

INTRODUCTION within host cells, generating intracellular bac-

terial communities (IBCs). Biofilms display
Klebsiella pneumoniae is a Gram-negative, increased resistance to antibiotics and host
encapsulated bacterium frequently found in immune defenses [16], and promote a favorable
hospital environments; it is primarily consid- environment for horizontal gene transmission
ered an opportunistic pathogen that colonizes [17].
human mucous membranes, including the gas- High antibiotic resistance is a hallmark in
trointestinal tract and the oropharynx. From K. pneumoniae infections. In addition to
these sites, K. pneumoniae can invade other tis- intrinsic resistance to antibiotics, K. pneumo-
sues and cause pneumonia, sepsis, meningitis, niae exhibits high levels of horizontal resis-
liver abscesses, urinary infections, among many tance transmission, mainly by conjugative
other diseases of high significance [1, 2]. plasmids, which allow the spread of resistance
K. pneumoniae can also cause infections in the to other microorganisms, of clinical impor-
community; in particular, the emergence of tance such as Acinetobacter baumannii, Pseu-
hypervirulent multidrug-resistant strains (MDR) domonas aeruginosa, Enterobacter cloacae and
in the community, such as extended spectrum Escherichia coli (members of the ESKAPE group)
beta-lactamase (ESBL)-producing and K. pneu- [18, 19]. Plasmid-associated carbapenemases,
moniae carbapenemase (KPC), is cause of great including KPC, NDM, IMP, VIM, and OXA-48
concern worldwide [3–7]. These strains display enzymes, are disseminated worldwide and
augmented capsule and siderophore produc- cause high rates of morbidity and mortality,
tion, as well as increased resistance to antimi- varying from 32% to 65% [20, 21]. KPC-pro-
crobials. Hypervirulent strains are more ducing K. pneumoniae is one of the first
prevalent in the community and affect indi- pathogens isolated from bloodstream infection
viduals of all age groups, causing diseases in in patients hospitalized in intensive care units
sites where the presence of K. pneumoniae is (ICU), a long hospital stay associated with
uncommon, such as endophthalmitis, menin- immunocompromised patients being the main
gitis, brain, epidural, and splenic abscess and risk factor to acquire this pathogen [10].
necrotizing fasciitis, in addition to increased The combination of increased and wide-
risk of other more common infections such as spread antibiotic resistance and the emergence
liver abscesses [8]. Of particular importance is of hypervirulent strains in community-ac-
the emergence of neonatal infections caused by quired infections place K. pneumoniae as a
K pneumoniae, which are associated with high pathogen of critical risk. According to a global
mortality in developing countries around the report by the World Health Organization
world [9–11]. According to the Burden of (WHO) [22], the antimicrobial resistance of
Antibiotic Resistance in Neonates from Devel- K. pneumoniae in severe healthcare-associated
oping Societies (BARNARDS) network, K. pneu- infections is around 50% worldwide. Despite
moniae is the main cause of neonatal sepsis in the recent approval of new antimicrobial
low- and middle-income countries, accounting options to treat KPC-producing K. pneumo-
for 10% of total sepsis deaths [12]. niae—especially ceftazidime-avibactam, a
Hyperproduction of polysaccharide capsule cephalosporin drug associated with a new
is the main virulence mechanism reported in beta-lactamase inhibitor avibactam—resistance
K. pneumoniae, contributing to immune evasion associated with KPC-3 and porin mutations or
and antimicrobial resistance [13]. Capsule pro- multiple carbapenemases production has been
duction is strictly associated with community- reported [23, 24].
acquired pneumonia and community-acquired Thus, there is an urgent need for effective
urinary infections by K. pneumoniae [14, 15]. In strategies to prevent K. pneumoniae infections.
addition, biofilm formation is an important Such formulations could prevent both nosoco-
virulence trait in K. pneumoniae; it can occur in mial and community-acquired infections,
both biotic and abiotic surfaces, as well as especially those associated with hypervirulent
strains. K. pneumoniae vaccines could target
Infect Dis Ther (2021) 10:2157–2175 2159

those at increased risk, including hospitalized Global Clinical Trials Data (globalclinicaltrials-
patients, immunocompromised individuals, data.com) search engines.
and newborns (either directly or through No exclusion date was determined for the
maternal immunization). The next sections selection of the publications because of the
summarize the current knowledge on K. pneu- limited sources of information related to the
moniae vaccines, grouped according to the advances in the development of K. pneumoniae
antigen used in the formulations. An overview vaccines. The search of the bibliography was
of the vaccine strategies under development conducted between September of 2020 and May
against K. pneumoniae is presented in Fig. 1. of 2021.
This article is based on previously conducted
studies and does not contain any new studies
METHODS with human participants or animals performed
by any of the authors.
In the current work, a literature review on
K. pneumoniae vaccine candidates was con-
ducted through data collected from the NCBI WHOLE CELL VACCINES/CELL
database, using the PubMed search engine LYSATES
(pubmed.ncbi.nlm.nih.gov/), with terms rela-
ted to the topic, in addition to each of the Whole cell K. pneumoniae vaccines have been
antigens evaluated and presented in this review. investigated alone or in combination with other
Clinical trial databases were used to search for enterobacteria. These formulations include live
studies with K. pneumoniae in progress; these attenuated bacteria, inactivated whole cells, and
included the US National Institutes of Health cell extracts.
(NIH) (clinicaltrials.gov) and European Clinical Mutant K. pneumoniae strains lacking the
Trial Register (EudraCT) (clinicaltrialsregis- housekeeping gene tonB were protective against
ter.eu) along with the ISRCTN (isrctn.com) and challenge when administered intraperitoneally
[25]. This mutant displayed reduced ability to
acquire iron from the extracellular environ-
ment; however, it was still able to secrete side-
rophores, which played a role in lung pathology
and bacteria dissemination in a mouse pneu-
monia model, as a result of the release of pro-
inflammatory cytokines interleukin-6 (IL-6),
CXCL1, CXCL2, IL-1b, and macrophage
inflammatory protein 3 alpha (MIP-3a) [26].
The mouse pneumonia model was also
employed to evaluate protection mediated by
an inactivated K. pneumoniae vaccine. Intranasal
vaccination induced the production of IgG and
IgM and protected against nasal challenge.
Interestingly, protection did not seem to be
mediated exclusively by antibodies against the
capsule; a serotype-independent, unidentified
Fig. 1 Vaccine strategies tested against Klebsiella pneumo-
niae: live attenuated vaccines based on genetically modified antigen was also protective in this model. IgG
bacteria; inactivated whole cell vaccines; outer membrane was also able to induce passive protection
vesicles containing numerous virulence factors; polysac- against intraperitoneal challenge [27].
charide and lipopolysaccharide (LPS)-based vaccines; pro- Mixed bacterial vaccines (MBVs) are formu-
tein-based vaccine (recombinant or purified from bacterial lations containing heat-killed pathogens. One
extracts); conjugate vaccines including PS–protein or of these formulations includes Haemophilus
LPS–protein fusions; ribosomal vaccines influenzae, Neisseria catarrhalis, Streptococcus
2160 Infect Dis Ther (2021) 10:2157–2175

pyogenes, K. pneumoniae, Staphylococcus aureus, HLA-DR in monocytes and polymorphonuclear

and Streptococcus pneumoniae strains. In an leukocytes (PMNs). Interferon-c (IFNc) and
infectious asthma study in children, immu- TNFa expression profile was also restored to
nization with the MBV reduced the frequency levels close to those showed in healthy indi-
and severity of infections, with its effect lasting viduals. No major adverse reactions were
for years. The findings were attributed to the reported up to 6 months following the study
hyposensitization generated by the low bacte- [33].
rial dosage, which was best tolerated by the Vaccines based on inactivated enterobacte-
participants [28]. However, in a clinical trial rial strains were able to protect against recurrent
with 95 institutionalized male individuals, the urinary tract infections (UTIs). Immunization
formulation failed to induce protection against with UromuneÒ, a sublingual spray containing
respiratory infections [29]. inactivated K. pneumoniae, E. coli, Proteus vul-
A double-blind clinical trial using tablets garis, and Enterococcus faecalis, provided protec-
containing either bacterial lysates of S. pneumo- tion against recurrent UTI in women in a
niae, H. influenzae, K. pneumoniae, Klebsiella phase II study. Adverse reactions related to the
ozaenae, S. aureus, S. pyogenes, Streptococcus viri- formulation were only reported with one par-
dans, and Neisseria catarrhalis (OM-85 BV) or ticipant, while small potential adverse reactions
ribosomal fractions derived from S. pneumoniae, occurred in seven participants [34]. A similar
S. pyogenes, H. influenzae, and K. pneumoniae study in Spain, with 784 patients, mostly
(D53) showed an induction of anti-K. pneumo- women, reported comparable results in the
niae antibody-producing cells in the tonsils of reduction of recurrent UTIs in the individuals,
children between 3 and 16 years old. The ribo- with minimum side effects reported [35]. The
somal formulation showed superior results specific immune responses induced by this for-
when compared to the whole cell vaccine [30]; mulation were not investigated in those studies.
however, the findings were later contested [31]. Another multivalent formulation to prevent
Ribosomal-based vaccines will be discussed in a UTIs, UrovacÒ, comprises E. faecalis, K. pneu-
separate section. moniae, Morganella morganii, Proteus mirabilis,
Another MBV is Dentavax, a formulation and six more E. coli strains administered locally,
composed of inactivated cells from K. pneumo- via vaginal suppositories. In phase II, random-
niae, S. pyogenes, S. aureus, Candida albicans, and ized, blinded, and placebo-controlled trials, the
Lactobacillus acidophilus, developed to prevent period between reinfections within the group
colonization and inflammation of the oral immunized with boosters was longer when
mucosa. In the study, 12 volunteers were given compared to the placebo and the vaccine plus
tablets containing the mixture, and systemic placebo dose group during the 6 months study
and mucosal responses were observed. An period. The strongest correlation was found in
increase in antibody production was detected in sexually active volunteers with recurrent uri-
serum (IgG) and saliva (IgA) from immunized nary infections. The vaccines were well toler-
subjects, as well as tumor necrosis factor alpha ated, and no severe adverse reactions were
(TNFa) production by peripheral blood lym- reported [36–38]. Oral administration of Uros-
phocytes [32]. Similarly, Respivax was designed timÒ—another formulation composed of E. coli,
to prevent respiratory infections and includes K. pneumoniae, P. mirabilis, and E. faecalis—re-
inactivated S. pneumoniae, N. catarrhalis, S. pyo- duced reinfections with these bacteria in 320
genes, H. influenzae type B, S. aureus, and participants with recurrent or chronic UTIs,
K. pneumoniae strains. The 25 participants pyelonephritis, or prostatitis, an effect that
enrolled in the clinical trial had recurrent correlated with increased secretory IgA and
infections and demonstrated reduced expres- phagocytosis [39]. Other MBVs include Ismi-
sion of immune cell receptors when compared genÒ as prophylaxis or as a coadjuvant in the
to healthy individuals. Immunization with the treatment of chronic obstructive pulmonary
MBV showed a protective immunomodulation, disease [40–44], and Lantigen B to prevent res-
inducing expression of TLR2, CD14, CD86, and piratory and ear infections and eczemas.
Infect Dis Ther (2021) 10:2157–2175 2161

Despite the improved health of the patients, no 59 years old that lasted throughout the study—
specific immunological parameters were evalu- 3 months for IgG and 2 months for IgM. Most
ated [45–47]. of the adverse reactions were local and only 13
Taken together, whole cell vaccines have of the 72 participants reported broader symp-
shown to be a promising approach to prevent toms, namely fever, headaches, and malaise.
respiratory and urinary infections by K. pneu- Passive immunization with anti-CPS IgG from
moniae; however, many of these formulations sera of the volunteers was also able to partially
display toxicity, which limits their widespread protect mice against sepsis in a burn wound
use. The length of the acquired protection and model [56]. A 24-valent CPS formulation from
the immunological mechanisms induced by K. pneumoniae and Klebsiella oxytoca was
these vaccines have not been fully administered in addition to an 8-valent
demonstrated. P. aeruginosa O antigen conjugated with Pseu-
domonas toxin A as a carrier protein. The IgG
Subunit Klebsiella pneumoniae Vaccines response against the different serotypes per-
sisted for 18 months after the first immuniza-
Capsular Polysaccharides tion [57]. The same formulations were
Recently, many reviews have explored the pos- administered alone or in addition to tetanus
sibility of a capsular polysaccharide-based vac- toxoid, in a clinical study of patients with acute
cine to prevent K. pneumoniae infections trauma. Although the number of volunteers was
[48–51]. The high immunogenicity and surface small (10 patients), the vaccines were well
exposure of capsules make them an interesting accepted, with minimum side effects, and the
vaccination strategy; however, the structural overall vaccine response was encouraging: all
variability and variations in geographic distri- but one PS (K35) induced a fourfold increase in
bution of serotypes limit the potential coverage antibody levels in at least 70% of the patients,
of these formulations [52–54]. Currently, 77 while seven patients reacted against 22 of 24 of
capsular serotypes (referred to as K polysaccha- the serotypes [58].
rides) have been identified in K. pneumoniae, A recent study investigating synthetic K2
with many other serotypes deriving from rear- capsular polysaccharides conjugated with
rangements in the cps and k loci (known as the diphtheria toxin (DT) demonstrated production
KL series) [51]. of specific anti-capsular antibodies in mice
Passive immunization using antibodies which displayed in vitro bactericidal activity
raised against the polysaccharide capsule (CPS) through complement system activation [59].
protected mice against sepsis and pneumonia. However, in vivo protection was not evaluated
Similarly, formulations containing membrane in that study.
polysaccharides (PS) were able to induce pro- Finally, Malachowa et al. recently developed
tection in animal models of infection [48]. A a pneumonia model to evaluate the protective
bioconjugation approach based on glycoengi- potential of vaccines based on purified capsule
neered E. coli expressing K. pneumoniae K1 and polysaccharide type 2 (CPS2) in cynomolgus
K2 antigens under regulation of rmpA (a regu- macaques. The vaccinated animals developed
lator of mucoid phenotype A) led to production anti-capsular antibodies which enhanced
of IgG against both glycans in mice and pro- in vitro serum bactericidal activity and
tected against lethal challenge with K1 and K2 opsonophagocytosis by neutrophils. They also
strains [55]. reported an increase in chemokines and
Phase I and II clinical trials were performed inflammatory cytokines such as IL-17A, which
using K. pneumoniae capsular polysaccharides, correlated with reduced bacterial loads in the
either alone or co-administered with a P. aerug- lungs [60].
inosa conjugate formulation. CPS from a K1 Polysaccharide-based vaccines have been
strain was able to induce IgM and IgG produc- successfully used to prevent disease by both
tion in healthy volunteers between 16 and Gram-negative and Gram-positive bacteria for
decades; their general high immunogenicity
2162 Infect Dis Ther (2021) 10:2157–2175

and external cellular localization make retaining the protection ability at a 100-fold
polysaccharides excellent immune targets. dose reduction. All formulations induced
Nevertheless, the high variability in capsule in vitro bacterial killing by macrophages [1].
polysaccharide composition greatly impacts Another pneumonia mouse model using LPS
vaccine coverage, while their inability to pro- showed that intranasal and intramuscular
mote T cell-dependent immune responses hin- administration of the vaccine reduced the bac-
ders vaccine efficacy, especially in young terial burden in the lungs; however, lung tissue
children, who are not able to respond to T cell- damage was reported [2]. The use of micropar-
independent antigens. This latter issue may be ticles of sodium alginate containing LPS had
overcome by polysaccharide conjugation to similar effects reducing the lung bacterial bur-
protein carriers, although this will greatly den; however, the tissue damage was higher
improve vaccine cost. This approach has been when compared to the free LPS administered
successfully used in the development of vacci- intranasally, intramuscularly, or intratracheally
nes against S. pneumoniae—a major human [3]
pathogen with over 100 capsular serotypes [61]. Rat pneumonia models were also used to
The great impact of pneumococcal conjugate assess the protective potential of LPS associated
vaccines—composed of 10 or 13 most prevalent with liposomes. This strategy was able to confer
capsule polysaccharides fused to protein carri- protection, reducing the lung bacterial load,
ers—in reducing disease burden across the while reducing the toxicity of LPS; whereas the
world [62] suggests that careful selection of the LPS alone and combined with liposomes were
most clinically relevant serotypes is a promising pyrogenic in a rabbit assay, the incorporation of
strategy to prevent disease by encapsulated LPS in the liposome nullified the pyrogenic
bacteria such as K. pneumoniae. activity of the formulation [4].
Detoxified LPS from E. coli complexed with a
Lipopolysaccharides Neisseria meningitidis outer membrane protein
The O antigen is an important virulence factor (OMP) increased survival against K. pneumoniae
in K. pneumoniae, directly related to survival and P. aeruginosa challenge and prevented sepsis
success after invasion of the host. There are 11 in neutropenic rats [64]. The formulation also
different O antigens found in K. pneumoniae, a induced higher IgG levels when compared to
reduced number when compared to the variety extracted and detoxified Brucella abortus LPS [5].
of polysaccharides that compose the capsule. Similarly, a liposome formulation composed
The O1, O2, O3, and O5 antigens are more of E. coli, P. aeruginosa, and Bacteroides fragilis
commonly present in the clinically relevant complete-core LPS and lipid A protected mice
strains, with 98% of the isolates being of ser- against lethal challenge. Sera from rabbits pre-
otypes O1 to O4 [51]. Since many K. pneumoniae viously immunized with the liposomal formu-
serotypes share a similar LPS backbone struc- lation reacted against multiple K. pneumoniae
ture, formulations targeting such structures can strains with different O antigen serotypes [65].
be promising strategies [63]. Anti-E. coli mAbs demonstrated comparable
Many different formulations based on LPS results, cross-reacting with K. pneumoniae lipid A
have been evaluated in different models of and Serratia marcescens LPS [66]. Purified anti-
K. pneumoniae infection. Extracted LPS was LPS or monoclonal antibodies against LPS anti-
evaluated against lobar pneumonia in mice and genic units granted immunity against systemic
was able to reduce lung burden following chal- and pulmonary infection in mice, reducing the
lenge; however, it was also pyrogenic in a rabbit bacterial burden in liver, spleen and lungs.
temperature assay. Interestingly, the reduction Cross-reaction of antibodies against LPS was
of carboxyl groups of LPS reduced the pyro- observed among strains containing different
genicity of the formulation and the conjugation types of O antigen [67–70].
of bovine serum albumin (BSA) with either Intramuscular or intraurethral administra-
lipid A or the extracted LPS inhibiting the tion of K. pneumoniae LPS was also protective in
increase in the rabbit’s temperature while a UTI mouse model. Both immunization routes
Infect Dis Ther (2021) 10:2157–2175 2163

led to a reduction in bacteria colonizing the structures, nucleic acids and proteins, such as
kidneys [71]. OMPs, cytoplasmatic proteins, and toxins
Besides being used as a vaccine antigen, LPS [75, 76]. OMVs are also related to immune sys-
has been evaluated as an adjuvant in conjuga- tem evasion and extracellular transport of bio-
tion with iron-regulated, cell surface proteins. molecules of interest [77]. Vaccine formulations
The conjugated vaccine reduced bacterial bur- containing OMVs and/or OMPs have been tes-
den in a pneumonia rat model and increased ted in different infection models.
bacterial phagocytosis [72]. Another conjugate K. pneumoniae OMVs were able to induce
vaccine included K. pneumoniae O1 coupled cellular immune responses in bronchial epithe-
with P. aeruginosa flagellin subunits FlaA and lial cells, with increased production of IL-1b, IL-
FlaB. The glycoconjugate induced high IgG 8, IL-6, and TNFa. [76]. Similar results were
levels against O antigen, and sera from immu- found for laryngeal epithelial cells, where OMVs
nized mice was able to reduce the bacterial induced IL-1b and IL-8. In an immunocompro-
burden in blood, liver, and spleen and mortality mised mouse model, the intratracheal inocula-
in mice. In rabbits, the conjugation of FlaA or tion of OMVs promoted pulmonary pathology,
FlaB to O1, O2, O3, or O5 increased the anti- suggesting a strong and potentially harmful
body response against O antigen when com- inflammatory response [75]. One of the reasons
pared to the group injected with the co- behind the immunogenicity of OMVs is the
administered antigens [73]. release of OMPs (OmpA and OmpC) inside the
Finally, a formulation including antigens vesicles, which contribute to the inflammatory
O1, O2, O3, and O5 coupled with the type III response [76]. Cytokine production by mouse
fimbriae adhesin MrkA composing a multiple- macrophages was also enhanced in the presence
antigen presenting system (MAPS) is under of OMVs containing secreted OmpK35 and
development by Affinivax [74]. OmpK36, with expression of TNFa, macrophage
Lipopolysaccharides are highly immuno- inflammatory protein 2, and granulocyte-mac-
genic molecules and important virulence fac- rophage colony-stimulating factor [78]
tors for K. pneumoniae. They are surface exposed In vitro assays demonstrated the production
and considerably less variable when compared of IL-6 and TNFa, along with overexpression of
to polysaccharides. Vaccine formulations uti- MHC II and CD86 in dendritic cells upon
lizing LPS or LPS fragments have been success- stimulation with OMVs [79]. The same study
fully tested in different models of infection. showed that the in vivo response to OMVs
However, the high toxicity of LPS is the main included a mixed Th1, Th2, and Th17 profile.
limiting factor related to this type of vaccine. Immunization with OMVs prevented septic
Detoxification protocols have been used to death after challenge. Protection was also
enhance vaccine safety, but a delicate balance achieved via passive immunization with sera or
between immunogenicity and toxicity must be splenocytes from immunized mice [79]. The
considered. Furthermore, LPS, such as polysac- addition of BSA nanoparticles to K. pneumoniae
charides, are T cell-independent antigens which OMVs increased the protective potential against
are not suitable for all age groups. This can be lethal challenge with KPC, which correlated
overcome by conjugation to protein carriers, with higher IgG production. Along with the
which have been shown to promote strong, in vivo protection, the combination boosted
lasting responses in children as previously the recruitment and activation of dendritic
described. cells, with overexpression of MHC II, CD40,
CD80, and CD86. In vitro assays showed
Outer Membrane Vesicles increased secretion of IL-6, TNFa, and IFNc by
Like many Gram-negative bacteria, K. pneumo- mouse macrophages after stimulation with the
niae produce outer membrane vesicles (OMVs), OMVs. IFNc levels were especially higher when
which constitute an important virulence factor compared to the non-BSA-paired OMV group
for this bacterium. OMVs comprise a lipid [80].
bilayer vesicle containing LPS, lipids, cell wall
2164 Infect Dis Ther (2021) 10:2157–2175

OMVs are a promising approach in vaccine between 3 and 16 years old, who had under-
development, since they induce specific adap- gone tonsillectomy. The tablet formulation
tive immune responses through the presence of named D53 stimulated specific anti-K. pneumo-
OMPs, and toxins, while displaying intrinsic niae antibody-producing cells at a higher level
adjuvant properties. OMVs have been used as than the bacterial lysate formulation OM-85
platforms for different vaccines, being particu- used in the same study [30]. As described in the
larly interesting as mucosal delivery systems OM-85 section, the results of the study were
[81]. Although OMVs are usually well tolerated later questioned [31].
and display low toxicity, the pulmonary Overall, the protective efficacy of ribosome-
pathology observed with intratracheal injection based vaccine formulations is controversial,
of K. pneumoniae OMVs raises safety concerns since many include surface protein contamina-
and suggests further studies are needed to tions, which may be major contributors to the
reduce undesirable side effects. Furthermore, protective responses. The ribosomes may act as
the complexity of OMV composition, with the adjuvants for these surface protein contami-
presence of multiple antigens in variable con- nants or, in some cases, carry mRNA for these
centrations makes it difficult to ensure consis- proteins which is then translated in the immu-
tency in the manufacturing procedures and nized host [82]. Ribosomal fractions also display
may affect quality control. immunomodulatory effects, with the induction
of T cell responses that contribute to B cell
Ribosomal-Based Formulations proliferation and increased antibody produc-
Vaccine formulations based on purified bacte- tion [87].
rial ribosomes or ribosomal fractions have been
investigated for many decades, and in general Protein-Based Formulations
promoted increased protection when compared Outer Membrane Proteins Various formula-
to whole cell vaccines [82]. tions based on recombinant OMPs have been
Purified ribosomes, proteins extracted from evaluated for their protective effect against
ribosomes, or extracted ribosomal RNA from K. pneumoniae infections. OmpA (also known as
K. pneumoniae showed protection in a systemic P40) has been suggested as a potential carrier in
lethal challenge model when injected subcuta- conjugated formulations, with similar efficiency
neously [83]. Passive immunization with spleen as tetanus toxoid [88]. When administered as a
cells was protective in a lethal challenge. How- carrier in combination with respiratory syncy-
ever, hyperimmune serum was not protective in tial virus subgroup A (RSV-A), it induced IgA,
this model, suggesting that the cellular response IgG1, and IgG2a production which were pro-
is the predominant protective mechanism tective against challenge [89]. These findings are
induced by these formulations [84]. supported by in vitro assays showing the
An aerosol formulation based on ribosomes upregulation in production of TNFa and nitric
of K. pneumoniae, S. pneumoniae, S. pyogenes, oxide in vaccinated mice, as well as increased
H. influenzae, in addition to membrane glyco- bacterial attachment and internalization by
proteins of K. pneumoniae, induced an increase human macrophages [90]. Similar results were
in IgG and IgM levels for at least 255 days after found after conjugation with a peptide derived
the first dose in the vast majority of the volun- from RSV attached to G protein and an H. in-
teers [85]. A similar formulation, Ribomunyl, fluenzae type B PS. Interestingly, immunization
was administered orally in patients with recur- with recombinant OmpA induced a mixed Th1/
rent infectious rhinitis. The treated group Th2-type response, including cytokines such as
showed reduced incidence compared to the IL-2, IL-5, IL-10, and IFNc [91].
placebo-treated individuals over a 6-month A vaccine including OmpA fused to OmpK36
study period [86]. In a similar, double-blind led to an increase in secretion of IL-2 and IFNc
clinical trial, ribosomal fractions derived from and protected mice from K. pneumoniae chal-
S. pneumoniae, S. pyogenes, H. influenzae, and lenge. Sera from immunized mice was able to
K. pneumoniae were given to 90 children prevent bacterial biofilm formation in vitro.
Infect Dis Ther (2021) 10:2157–2175 2165

Despite the encouraging results, the induced In sum, the literature indicates that recom-
antibodies showed cross-reactivity with OMPs binant OMPs are promising vaccine candidates
present in other Enterobacteriaceae strains, against K. pneumoniae, alone or combined with
which could lead to undesirable effects on the other antigens. Owing to their adjuvant effects,
host microbiota [92]. OmpK17 and OmpK36 these proteins can also act as immunomodula-
were able to protect against lethal challenge and tory agents in cancer vaccines. However, the
induced IgG1 and IgG2a; a chimera formulation cross-reactivity with molecules expressed by
of both proteins induced a Th2-type response other enterobacteria suggest a possible interfer-
with strong IgG1 production [93]. ence with the host microbiota, which could be
Similarly to the results using recombinant overcome with bioinformatic tools to select
OMPs, mouse immunization with a DNA vac- specific immunogenic epitopes that are not
cine including ompA and ompK36 was capable of shared by commensal bacteria.
inducing an increase in IgG and interleukins,
and conferred protection against lethal chal- Fimbriae Subunits In K. pneumoniae, two
lenge [94]. Extracted, LPS-free OMPs from a main types of fimbriae are found, type I and
CPS-deficient strain protected against lethal type III. The fim gene cluster is responsible for
challenge with a wild-type strain, and a similar type I fimbriae, which are related to adhesion to
effect was observed with passive immunization the epithelium and to mucous membranes [2].
using OMP-induced antibodies [95]. The fimbriae components encoded by the mrk
In silico analyses have also demonstrated gene cluster are responsible for the type III
that OMPs are promising candidates for a Kleb- fimbrial subunits, which are essential for adhe-
siella vaccine [96]. A study using reverse vacci- sion and biofilm formation on biotic and abi-
nology selected candidates for heterologous otic surfaces [101, 102]. Other fimbriae clusters
expression from K. pneumoniae antigens with have been identified [103], although some of
predicted T and B cell epitopes. The epitopes these new clusters are part of the accessory
were fused in a chimera, using cholera toxin genome, and their distribution is species-de-
subunit B as adjuvant. Among the epitopes, pendent [2].
those derived from OmpA and PhoE (an outer Type I fimbriae extracted from K. pneumoniae
membrane porin) were among the most and type III fimbriae from K. oxytoca were eval-
promising candidates; however, to date, the uated as protein carriers in conjugation with
formulation was still not tested in vivo [97]. E. coli endotoxin. Although the level of TNFa
Proteomic analysis showed that OmpN and secreted by murine fibroblasts was similar to
CusC are interesting vaccine candidates, since that induced by endotoxin alone, there was a
they are highly conserved among different slight increase in the production of IL-6 and IFN
strains and predicted to be antigenic, while in the presence of both fimbriae conjugates, in
displaying no homology to proteins found in A549 and 7TD1 cells, respectively [104].
members of the human microbiota [98]. In K. pneumoniae, immunization using
OmpA was also proposed as an adjuvant in recombinant MrkD or MrkD-derived peptides
melanoma vaccines, which have undergone was capable of activating CD4? T lymphocytes,
phase I clinical trials [99]. Another study eval- with production of IL-4 and IFNc [105]. In
uated OmpA as a carrier protein following another study, immunization with recombi-
conjugation to S. pneumoniae CPS 14 and 19. nant MrkA or anti-MrkA monoclonal antibodies
The addition of OmpA to the pneumococcal induced protection in mice against pneumonia.
polysaccharides increased IgM and IgG pro- These monoclonal antibodies reduced in vitro
duction, mainly IgG1 and IgG3, against biofilm formation and cell adhesion, cross-re-
S. pneumoniae CPS 14 and 19 [100]. Passive acted with strains of different serotypes, and
immunization with anti-OmpA-CPS19 anti- promoted bacterial opsonophagocytosis
bodies was partially protective against pneu- [106, 107].
mococcal challenge. Immunization using purified type III fim-
briae from K. pneumoniae was protective in a
2166 Infect Dis Ther (2021) 10:2157–2175

mouse pneumonia model, using a low dose of disease pathogenesis makes them ideal targets
bacteria. Protection was correlated with specific for vaccine development, and many have been
IgG production; however, other protection investigated in animal models.
parameters were not evaluated. Passive immu- Purified heat-stable K. pneumoniae entero-
nization was also achieved via administration of toxin was able to protect against lethal chal-
sera from hyperimmune animals [108]. lenge, with similar results to those induced by
In E. coli, fimbrial subunits have been E. coli enterotoxin in rats [122]. Toxoids from
extensively investigated as potential vaccine K. pneumoniae derived from endogenous cyto-
candidates. Immunization using E. coli type I toxin I promoted protection in a systemic
fimbriae subunits/adhesins showed promising model of infection in mice. Among the toxoids,
results against UTIs in various animal models KCT-I and PBE provided protection against
[109–117]. FimH was also tested as a protein K. pneumoniae and K. oxytoca strains. In female
carrier in a chitosan nanoparticle, DNA vaccine rabbits, the immunization induced elevated IgG
against coxsackievirus. The addition of FimH levels, which were later also found in their off-
increased the survival of mice and induced the spring’s serum. Similarly, passive immunization
production of fecal sIgA and serum IgG, in using colostrum from previously immunized
addition to stimulating T cell proliferation in animals was protective to newborn rabbits, after
spleen and lymph nodes. Increase in IFNc in lethal challenge [123], demonstrating a role for
splenocytes was also observed, along with the antibody response in this model.
higher specificity of cytotoxic T cells. mRNA Toxins have been traditionally used as
overexpression of CD80, CD86, and MHC II was human vaccines. The DTaP vaccine includes
reported in dendritic cells [118]. tetanus and diphtheria toxoids combined with
Generally, fimbriae components have been acellular pertussis, and is administered to chil-
shown to be promising candidates for inclusion dren in many countries, with booster doses of
in vaccines. Their contribution to important DT recommended for adults. Toxins make
virulence traits, such as biofilm formation and excellent vaccines owing to their high
adhesion to host cells, in addition to their sur- immunogenicity and pivotal role in disease
face exposure, reinforces their potential to elicit pathogenesis. The main concern is their
protective immune responses. However, since intrinsic toxicity, which must be abrogated to
most studies were conducted using E. coli, fur- allow their safe utilization. Several detoxifica-
ther work is needed to assess the protective role tion protocols, including physical treatments,
of recombinant fimbriae subunits in different chemical inactivation, and site-directed muta-
models of K. pneumoniae infection. Owing to genesis, may be employed to produce toxoids—
the high similarity between these two species of the non-toxic yet immunogenic form of the
enterobacteria, an approach targeting the toxins.
highly conserved fimbriae genes of K. pneumo-
niae could provide interesting results. Other Proteins Siderophores and membrane
receptors are interesting vaccine candidates
Toxins K. pneumoniae (along with other owing to their cellular location—which allows
members of the ESKAPE group) produce and prompt recognition by the host immune sys-
secrete toxins and anti-toxins, especially the tem—and contribution to virulence. Therefore,
hypervirulent strains. These biomolecules are different molecules involved in nutrient/ion
related to virulence, including an increased transport and environmental sensors have been
survival in the presence of antimicrobials [119]. proposed as vaccine candidates.
One toxin in particular, colibactin, is genotoxic Siderophore receptor protein (SRP) was able
and able to hinder the host cell cycle [120]. to protect against bacterial bovine mastitis
Such deleterious activity has been implicated in caused by Klebsiella, as well as other relevant
colorectal cancer development and neonatal coliforms [124]. FepA, a protein related to side-
systemic infections, namely sepsis and menin- rophore activity, was proposed as a potential
gitis [121]. The important role of such toxins in candidate after immunoproteomic analysis of
Infect Dis Ther (2021) 10:2157–2175 2167

infected patients [94]. FepB, a enterobactin Subsequently, purified IgG showed the ability to
transporter, was chosen among many conserved prevent iron acquisition from E. coli isolates
proteins as a potential candidate using reverse [131]. The ferric enterobactin receptor FepA
vaccinology [98]. A conjugated vaccine com- exhibited similar iron acquisition blocking to
posed of iron-regulated, cell surface proteins FecA, with synergistic activity when combined
and LPS as an adjuvant induced antibodies with [132]. In a cow mastitis model induced via local
increased phagocytic ability and reduced the inoculation of E. coli, though FecA was deemed
bacterial burden in a pneumonia rat model [72]. immunogenic, it had no effect on the clinical
FyuA is a conserved yersiniabactin receptor manifestations [133].
of Enterobacteriaceae, directly related to side- Surface environmental sensors, proteins
rophore expression. Immunization with FyuA involved in nutrient uptake, or molecule secre-
prevented pneumonia in a mouse model, an tion systems are interesting vaccine candidates,
effect that correlated with an early increase in because the induction of immune responses
IL-17, IL-1b, and TNFa in the lungs after the first that limit their functions will affect the patho-
day of infection, followed by a rapid decrease on gen’s ability to colonize the host. In addition to
the next day. Serum from the immunized group the well-studied siderophores and transport
also promoted an increase in bacterial phago- systems, bioinformatics has provided many new
cytosis and higher bactericidal activity [125]. potential vaccine candidates on the basis of
Antigenome analyses revealed proteins con- their predicted cell localization and function.
served among different serotypes as potential Although further studies are needed to confirm
vaccine candidates, including RecX, RecO, which of those molecules provide the best,
YhiN, and YfhM. DNA vaccines including genes long-term immunity, data from other patho-
from these antigens were effective in a sepsis gens such as N. meningitidis has proven the
model [126]. Colicin I, a bacteriocin receptor, is success of this new technology in vaccine
predicted to be highly immunogenic and sug- development.
gested as a potential target in vaccine formula-
tions, following an immunoproteomic analysis
of a clinical, drug-resistant K. pneumoniae strain; CLOSING REMARKS
however, the protective potential of the protein
was never evaluated [127]. Another proteomic- Infections caused by K. pneumoniae represent a
based analysis suggested the zinc transporter major threat to public health worldwide. In the
ZnuA, the ribonuclease H1, the TehB methyl- past, K. pneumoniae was mainly considered an
transferase, and two more hypothetical proteins opportunistic pathogen; however, the rapid
(WP_002918223 and WP_002892366) as candi- increase in community-acquired Klebsiella
dates for a vaccine formulation, based on con- infections, including multidrug-resistant strains
servation among strains and immunogenicity [3–7], has placed this bacterium amongst the
prediction [98]. top pathogens. Therefore, the development of
Recently, another candidate, the predicted vaccines against K. pneumoniae has become a
adhesin YidR, has been tested in different ani- priority in many countries [49].
mal models. The protein was able to protect As previously shown for several pathogens,
mice against lethal challenge [128] and vaccination is an effective way to prevent dis-
improved bovine milk production via protec- ease, especially when considering those more
tion against Klebsiella and E. coli infections susceptible—the elderly, children, and
[129]. In E. coli bovine mastitis isolates, the fer- immunocompromised individuals. Several
ric citrate receptor FecA was found to be over- approaches have been explored to prevent
expressed. Serum from rabbits immunized with K. pneumoniae infections, including whole cells
FecA showed binding to E. coli, and also to and cell extracts, ribosomal vaccines, and viru-
K. pneumoniae mastitis isolates in an immuno- lence factor including OMVs, capsular polysac-
blot assay [130]. Cow immunization with FecA charides, LPS, and proteins (Fig. 1). Some of
was able to induce high titers of IgG.
2168 Infect Dis Ther (2021) 10:2157–2175

these formulations have been successful and ACKNOWLEDGEMENTS

progressed to clinical trials.
While all vaccine approaches have limita-
tions—these being the increased reactogenicity Funding. Lucas Assoni has a scholarship from
of whole cell vaccines or the lower immuno- Coordenação de Aperfeiçoamento de Pessoal de
genicity of subunit formulations—there is Nı́vel Superior (CAPES) (grant 88887.601281/
increasing evidence that K. pneumoniae vaccines 2021-00). No Rapid Service Fee was received by
can be a reality within the next decades. Par- the journal for the publication of this article.
ticularly, the use of conjugate vaccines includ-
ing capsular polysaccharides and/or O antigens Authorship. All named authors meet the
fused to protein carriers is an alternative with International Committee of Medical Journal
great potential. The careful selection of the Editors (ICMJE) criteria for authorship for this
most clinically relevant capsular and article, take responsibility for the integrity of
O serotypes is key to developing effective vac- the work, and have given their approval for this
cines with broad coverage. Also, since essential version to be published.
proteins have a tendency to be conserved
within the species, the inclusion of K. pneumo- Author Contributions. The authors con-
niae surface proteins as carriers could prevent tributed to the present work as follows. Litera-
serotype replacement in the long term [134], ture search (LA and MD), Original draft
while increasing the protective efficacy of the formulation (LA, TRC and MD). Review of the
formulations. manuscript (RG, TRC and MD). Conceptualiza-
The development of subunit vaccines has tion of the figure (LA and MD). Overall super-
been greatly impacted in recent years by new vision of the work (TRC and MD).
techniques to identify and validate potential
candidates. The antigenome strategy, combin- Disclosures. Lucas Assoni, Raquel Gir-
ing informatic tools with knowledge on disease ardello, Thiago Rojas Converso and Michelle
pathogenesis, has provided a great number of Darrieux have nothing to disclose.
new potential vaccine targets to be explored.
These molecules can be tested alone or com- Compliance with Ethics Guidelines. This
bined to generate multicomponent vaccines to article is based on previously conducted studies
tackle different disease stages or niches. and does not contain any new studies with
In conclusion, there is an urgent need for human participants or animals performed by
effective K. pneumoniae vaccines that are able to any of the authors.
protect against the increasing numbers of mul-
Open Access. This article is licensed under a
tidrug-resistant and hypervirulent strains found
Creative Commons Attribution-Non-
in the community. Given the complexity of
Commercial 4.0 International License, which
K. pneumoniae infections, it is plausible to
permits any non-commercial use, sharing,
assume that different formulations will be gen-
adaptation, distribution and reproduction in
erated to prevent diverse forms of disease.
any medium or format, as long as you give
Multicomponent conjugate vaccines comprise
appropriate credit to the original author(s) and
the most promising strategy to provide long-
the source, provide a link to the Creative
term, broad vaccine coverage. The use of
Commons licence, and indicate if changes were
recombinant systems allows safe production of
made. The images or other third party material
vaccinal antigens with high yields, while the
in this article are included in the article’s
combination of genomic/proteomic analysis
Creative Commons licence, unless indicated
and better understanding of disease pathogen-
otherwise in a credit line to the material. If
esis refines the selection of new candidates.
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Infect Dis Ther (2021) 10:2157–2175 2169

permitted use, you will need to obtain permis- 10. Justo-da-Silva LH, De-Azeredo AN, Bueno AC, et al.
sion directly from the copyright holder. To view Diversity of clonal types of Klebsiella pneumoniae
causing infections in intensive care neonatal
a copy of this licence, visit http:// patients in a large urban setting. Braz J Microbiol.
creativecommons.org/licenses/by-nc/4.0/. 2019;50(4):935–42.

11. Sankar MJ, Agarwal R, Deorari AK, Paul VK. Sepsis in

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